Method and compositions for inhibiting or preventing adverse effects of oral antibiotics

ABSTRACT

This invention provides, in part, various compositions and methods for protecting the gastrointestinal microbiome from antibiotic disruption.

RELATED APPLICATIONS

The present application claims priority to US Provisional ApplicationNos. 62/096,202, filed Dec. 23, 2014, and 62/256,994, filed Nov. 18,2015, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to, in part, various compositions and methods forprotecting the gastrointestinal microbiome from antibiotic disruption.

BACKGROUND

The human microbiome is proving to be a vital component in both humanhealth and disease. This is particularly true of the gastrointestinal(GI) tract, which houses over one thousand distinct bacterial speciesand an estimated excess of 1×10¹⁴ microorganisms, and appears to becentral in defining human host health status. For example, themicrobiome of the GI tract underlies central processes of nutrientcapture and metabolism; however, disruption of this microbiome is alsobelieved to be causative of a number of disorders.

Indeed, antibiotics, often a frontline therapy to prevent deleteriouseffects of microbes on human health, can induce disruption in themicrobiome, including in the GI tract, and lead to further disease. Forinstance, it is often necessary to administer oral antibiotics for thetreatment of infections. However, residual oral antibiotics beyond whatis needed for eradication of an infection can alter the ecologicalbalance of normal intestinal microbiota in the gut and lead to furtherdisease.

Therefore, there is a need for agents that prevent microbiome disruptionby oral antibiotics while not reducing or eradicating the beneficialanti-infective effects of these antibiotics in a subject.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides compositions and methods forprotecting the gastrointestinal microbiome of a subject. In one aspect,methods for protecting the microbiome of the GI tract are provided inwhich an effective amount of a pharmaceutical composition comprising abeta-lactamase is administered to a subject who is undergoing treatmentof has recently undergone treatment with an oral antibiotic, wherein thebeta-lactamase is capable of deactivating the oral antibiotic. In anembodiment, the beta-lactamase does not substantially interfere with theplasma levels of a systemically absorbed orally administered antibiotic.In another embodiment, the beta-lactamase deactivates excess oralantibiotic residue excreted into the GI tract. In some embodiments, thebeta-lactamase deactivates residual active antibiotic that is notabsorbed from the GI tract after an oral dose or is returned in activeform to the intestinal tract from the systemic circulation. In certainembodiments, an initial and/or adjunctive therapy may be administered tothe subject. The initial and/or adjunctive therapy may be one or more ofmetronidazole, vancomycin, fidaxomicin, rifaximin, charcoal-basedbinder/adsorbent, fecal bacteriotherapy, probiotic therapy, and antibodytherapy. In certain embodiments, the subject may have previouslysuffered from a microbiome-mediated disorder or may present withsymptoms of recurrence of a microbiome-mediated disorder.

In various embodiments, the methods of the invention treat or prevent amicrobiome-mediated disorder, such as an antibiotic-induced adverseeffect, Clostridium difficile (C. difficile) infection, C.difficile-associated disease, ulcerative colitis, Crohn's disease, andirritable bowel syndrome. In an embodiment, the methods of the inventionmaintain the normal intestinal microbiota of a subject. For instance, insome embodiments, the methods of the invention maintain a healthybalance (e.g. a healthy ratio and/or healthy distribution) of intestinalmicrobiota of a subject. In another embodiment, the methods of theinvention treat or prevent the overgrowth of one or more pathogenicmicroorganisms in the GI tract. In a further embodiment, the methods ofthe inventions find use in treating or preventing a nosocomial infectionand/or a secondary emergent infection.

In various embodiments, the beta-lactamase is formulated for GI tractdelivery. For example, the beta-lactamase may be enteric coated. In anembodiment, the beta-lactamase is formulated for release in a locationin the GI tract in which it deactivates residual or excess oralantibiotic (e.g. residual active antibiotic that is not absorbed fromthe GI tract after an oral dose or is returned in active form to theintestinal tract from the systemic circulation). In another embodiment,the beta-lactamase is formulated for release in a location in which itprevents a microbicidal activity of the residual or excess oralantibiotic. In a further embodiment, the beta-lactamase is formulatedfor release in a location in the GI tract in which it does notsubstantially interfere with the systemic activity of the orallyadministered antibiotic. In another embodiment, the beta-lactamase isformulated for release in a location in the GI tract that is distal tothe release and absorption of the orally administered antibiotic. Invarious embodiments, the beta-lactamase is formulated for substantiallyuniform dissolution in the area of release in the GI tract. In stillfurther embodiments, the beta-lactamase is formulated formicroorganism-based release in the GI tract.

In various embodiments, methods of the present invention providecombination therapy including beta-lactamase and one or more additionaltherapeutic agents. In an embodiment, a subject is administered with abeta-lactamase inhibitor that releases in the GI tract proximal to thebeta-lactamase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a timeline of piglet dosing for the study of Example 1.

FIG. 2 shows amoxicillin levels in the pig serum from the study ofExample 1.

FIG. 3 shows strain relative abundance percent similarity from thesequencing analysis of Example 1. Amoxicillin alone is displayed as thesolid line, and Amoxicillin+P3A or SYN-004 (synonyms for the sameenzyme) is displayed as the dashed line.

FIG. 4 shows strain relative abundance percent similarity boxplot fromthe sequencing analysis of Example 1. Amoxicillin alone is displayed asthe gray box, and Amoxicillin+SYN-004 is displayed as the white box. Theboxplot displays the median (line), the quartiles (box) and the range(vertical lines).

FIG. 5 shows strain abundance heat map from the sequencing analysis ofExample 1.

FIG. 6 shows the processing scheme for spray layered multiparticulatesas described in Example 2.

FIG. 7 depicts characterization of the enteric-coated SYN-004 particlesthat release at pH 6.2. Particles coated with Eudragit L100, EudragitS100, and triethyl citrate at a ratio of 72.7/18.2/9.1 werecharacterized based on the average coat thickness vs estimated coatweights (panel A) and the mass fraction vs the particle size (panel B).

FIG. 8 shows scanning electron microscope images of the enteric-coatedSYN-004 particles that release at pH 6.2. Particles coated with EudragitL100, Eudragit S100, and triethyl citrate at a ratio of 72.7/18.2/9.1and at different coat weights, i.e., 25%, 30%, and 35%, were subjectedto scanning electron microscopy. The top panels display the 50×magnification for particle size characterization, and the lower panelsdisplay the 700× magnification of particle cross sections (n=6) for eachcoating % to allow determination of the coating thicknesses.

FIG. 9 shows scanning electron microscope images of the enteric-coatedSYN-004 particles that release at pH 6.2. Particles coated with EudragitL100, Eudragit S100, and triethyl citrate at a ratio of 72.7/18.2/9.1 ata 35% coat weight were subjected to scanning electron microscopy. Thepanels, from left to right, display the 50× magnification for particlesize characterization, 250× magnification for surface uniformityanalyses, 50× magnification of a particle cross section, and a schematicdiagram of a particle displaying the three layers.

FIG. 10 depicts characterization of the enteric-coated SYN-004 particlesthat release at pH 6.7. Particles coated with Eudragit L100, EudragitS100, and triethyl citrate at a ratio of 30/60.9/9.1 were characterizedbased on the average coat thickness vs estimated coat weights (panel A)and the mass fraction vs the particle size (panel B).

FIG. 11 shows scanning electron microscope images of enteric-coatedSYN-004 particles that release at pH 6.7. Particles coated with EudragitL100, Eudragit S100, and triethyl citrate at a ratio of 30/60.9/9.1 andat different coat weights, i.e., 25%, 30%, and 35%, were subjected toscanning electron microscopy. The top panels display the 50×magnification for particle size characterization, and the lower panelsdisplay the 400× magnification of particle cross sections (n=6) for eachcoating % to allow determination of the coating thicknesses.

FIG. 12 shows scanning electron microscope images enteric-coated SYN-004particles that release at pH 6.7. Particles coated with Eudragit L100,Eudragit S100, and triethyl citrate at a ratio of 30/60.9/9.1 at a 35%coat weight were subjected to scanning electron microscopy. The panels,from left to right, display the 50× magnification for particle sizecharacterization, 1000× magnification for surface uniformity analyses,50× magnification of a particle cross section, and a schematic diagramof a particle displaying the three layers.

FIG. 13 depicts the osmotic rupture of coated particles. Particles with10% or 11.5% osmotic coat weights with cure temperatures of 50° C. or60° C., and cure times of 2, 5, and 8 hours were compared. The indicatedpellets were added to a 50 mM KH₂PO₄ pH 6.2 buffer at room temperaturewithout stirring and images of the pellets were taken every 5 minutesover 7 hours to evaluate particle disruption. Particle disruptionincluded visible coating changes and significant deformation of theparticles. The samples included: 10% coating, cure at 60° C. for 2 hour(diamonds); 11.5% coating, cure at 50° C. for 2 hour (triangles); 11.5%coating, cure at 50° C. for 5 hour (squares); 11.5% coating, cure at 50°C. for 8 hours (Xs); and 11.5% coating, cure at 60° C. for 2 hours(asterisks).

FIG. 14 depicts the osmotic rupture of coated particles. Particles with7.3%, 9.1%, 10%, 11.4%, or 13.5% osmotic coat weights were compared. Theindicated pellets were added to a 50 mM KH₂PO₄ pH 6.2 buffer at roomtemperature without stirring and images of the pellets were taken every5 minutes over 10 hours to evaluate particle disruption. Particledisruption included visible coating changes and significant deformationof the particles. The samples included: 7.3% coating (triangles); 9.1%coating (squares); 10% coating (diamonds); 11.4% coating (asterisks);and 13.5% coating (Xs).

FIG. 15 shows the osmotic rupture of coated particles. Photos ofparticles with 10% or 13.5% osmotic coat weights are displayed. Theindicated pellets were added to a 50 mM KH₂PO₄ pH 6.2 buffer at roomtemperature without stirring. The top panels display the 10% coat weightparticles at 0, 2, and 4 hours of soaking. The bottom panels display the13.5% coat weight particles at 0, 6, and 8.5 hours of soaking.

FIG. 16 depicts characterization of the osmotic rupture SYN-004particles. The particles were characterized based on the average coatthickness vs estimated coat weights (panel A) and the mass fraction vsthe particle size (panel B).

FIG. 17 shows scanning electron microscope images of the osmotic ruptureSYN-004 particles. The osmotic rupture particles of different coatweights, 10%, 11.5%, and 13.5% were subjected to scanning electronmicroscopy. The top panels display the 50× magnification for particlesize characterization, and the lower panels display the 1000× or 400×magnification of particle cross sections (n=10) for each coating % toallow determination of the coating thicknesses.

FIG. 18 shows scanning electron microscope images of the osmotic ruptureSYN-004 particles. The 13.5% coating weight osmotic rupture particleswere subjected to scanning electron microscopy. The panels, from left toright, display the 50× magnification for particle size characterization,250× magnification for surface uniformity analyses, 50× magnification ofa particle cross section, and a schematic diagram of a particledisplaying the three layers.

FIG. 19 shows evaluations of cure time and temperature on enzymeactivity for osmotic rupture SYN-004 particles. The SYN-004 coatedsucrose pellets were coated with a sweller layer and then coated withthe osmotic rupture layer. The osmotic layer required a curing step.Cure temperatures of 50° C. or 60° C., and cure times of 0, 2, 5, and 8hours were evaluated. Pellets were added to a pH 6.8 potassium phosphatebuffer and stirred overnight to ensure removal of the entire coating.Aliquots of the buffer were analyzed for SYN-004 biological activityusing the CENTA chromogenic microtiter plate assay. Activity isdisplayed as % of theoretical activity based on the amount of SYN-004protein present in each formulation. Uncoated (SYN-004 pellet startingmaterial) is displayed as the triangle. 50° C. curing temperature isdisplayed as the diamond, and 60° C. is displayed as the square.

FIG. 20 provides a schematic representation of the criteria for choosinga modified-release formulation of SYN-004 for oral delivery with oralantibiotics. The desired outcome is to not interfere with antibioticabsorption from the intestinal track to maximize antibioticbioavailability, and to degrade antibiotic that is in the intestinaltract prior to causing damage to the microflora.

FIG. 21 depicts the SYN-004 pellet dissolution profile. The threeSYN-004 formulations (7.5 mg active) were incubated in 0.01N HCl (pH2.0) for 2 hours, pH 5.5 for 2 hours, and pH 6.5 up to 24 hours. Sampleswere tested for protein concentration by measuring absorbance at 280 nm(solid lines) and SYN-004 biological activity using the CENT chromogenicassay (dotted lines). The formulations were enteric pH 6.2, enteric pH6.7, and osmotic as described in Example 2.

FIG. 22 depicts the SYN-004 pellet dissolution profile compared tooriginal SYN-004 formulation. The original (enteric, pH 5.5) and thethree SYN-004 formulations (7.5 mg active) were incubated in 0.01N HCl(pH 2.0) for 2 hours, pH 5.5 for 2 hours, and pH 6.5 up to 24 hours.Samples were tested for protein concentration by measuring absorbance at280 nm (solid lines) and SYN-004 biological activity using the CENTchromogenic assay (dotted lines). The formulations were SYN-004 original(SynBio enteric, pH 5.5), enteric pH 6.2, enteric pH 6.7, and osmoticasdescribed in Example 2.

FIG. 23 depicts the capsule vs pellet dissolution profiles for the threeSYN-004 formulations. Capsules or pellets (cores) of the three SYN-004formulations were incubated in 0.01N HCl (pH 2.0) for 2 hours, pH 5.5for 2 hours, and pH 6.5 for up to 24 hours. Samples were tested forprotein concentration by measuring absorbance at 280 nm. Theformulations were Enteric pH 6.2, left panel, Enteric pH 6.7, middlepanel, and Osmotic, right panel.

FIG. 24 shows a timeline of piglet dosing. Animals received SYN-004 for9 days starting on Day 0. Animals received oral amoxicillinfor 7 daysstarting on Day 1. Stool was collected at 5 times, Day −7, Day −4, Day4, Day 8, and Day 9. Blood was collected at 3 times during Day 2.

FIG. 25 shows various formulation approaches of the invention.

FIG. 26 shows various formulation approaches for segregating antibioticand/or beta lactamase inhibitor and beta lactamase release.

FIG. 27 shows various combination dosage forms.

FIG. 28 shows various microparticulate dosage forms.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery thatbeta-lactamases can protect the gastrointestinal microbiome of a subjectwho is undergoing treatment or has undergone treatment with an oralantibiotic. Administration of oral antibiotics often disrupts theecological balance of normal intestinal microbiota due to residualunabsorbed antibiotics being excreted into the intestines (e.g., thedistal small intestine and/or the large intestine). Beta-lactamasesinactivate the unabsorbed antibiotics in the GI tract thereby restoringand/or maintaining the normal intestinal microbiota and preventing anyovergrowth of potentially pathogenic microorganisms.

In some aspects, the present invention is based, in part, on thediscovery that one or more beta-lactamases can be formulated to releasein one or more locations within the GI tract at which the beta-lactamaseinactivates (e.g. hydrolyzes) an orally delivered beta-lactam antibioticand, in doing so, protects the microbiome, but the beta-lactamase doesnot interfere with intestinal absorption of the oral antibiotic and,accordingly, does not interfere with systemic blood or plasma levels ofthe oral antibiotic. The invention further identifies the location ofsuch beta-lactamase release or activation with preferred locations beingin the ileum or cecum. By way of illustration, in some embodiments, thefollowing two approaches may be employed separately or in combination:utilization of formulations designed to release beta-lactamase at thedesired location in the GI tract and combining the antibiotic with anoral beta-lactamase inhibitor. In the latter, in some embodiments, thebeta-lactamase inhibitor tracks with the beta-lactam antibiotic suchthat both are available for absorption in the proximal small intestine.The beta-lactamase inhibitor serves to protect the beta-lactamantibiotic from the beta-lactamase in the proximal small intestine. Theantibiotic and the inhibitor are then both absorbed into the bloodstreamand thereby removed from the proximal small intestine. As theconcentration of inhibitor decreases in the small intestine, thebeta-lactamase becomes active. Any residual or excess antibiotic thatremains in the intestine or reenters with the bile will is inactivatedprior to encountering the colonic microbiome.

Beta-Lactamases and Pharmaceutical Compositions

The present invention is directed, in part, to pharmaceuticalcompositions, formulations, and uses of one or more beta-lactamases. Asused herein, a beta-lactamase refers to an enzyme, which deactivatesbeta-lactams. For example, the beta-lactamase may deactivate abeta-lactam by hydrolysis (e.g. hydrolysis of residual or excessantibiotic). Hydrolysis of the amide bond of the beta-lactam ring by thebeta-lactamase makes an antimicrobial agent such as an antibioticbiologically inactive.

In various embodiments, the present invention is directed tocompositions including one or more beta-lactamase enzyme of class EC3.5.2.6. In some embodiments, the beta-lactamase is a group 1, 2, 3, or4 beta-lactamase, in accordance with the functional classificationscheme proposed by Bush et al. (1995, Antimicrob. Agents Chemother. 39:1211-1233; the entire contents of which are incorporated herein byreference). Without wishing to be bound by theory, Group 1beta-lactamases include cephalosporinases that are not well inhibited byclavulanic acid; Group 2 includes penicillinases, cephalosporinases andbroad-spectrum beta-lactamases that are generally inhibited by activesite-directed beta-lactamase inhibitors; Group 3 includesmetallo-beta-lactamases that hydrolyze penicillins, cephalosporins andcarbapenems, and that are poorly inhibited by almost allbeta-lactam-containing molecules; and Group 4 includes penicillinasesthat are not well inhibited by clavulanic acid.

In some embodiments, the beta-lactamase is a class A, B, C, or Dbeta-lactamase, in accordance with the Ambler classification whichdivides beta-lactamases based on their amino acid sequences (Ambler1980, Philos Trans R Soc Lond B Biol Sci. 289: 321-331; the entirecontents of which are incorporated herein by reference). Without wishingto be bound by theory, classes A, C, and D beta-lactamases includeevolutionarily distinct groups of serine beta-lactamases, and class Binclude the zinc-dependent (“EDTA-inhibited”) beta-lactamases (seeAmbler R. P. et al., 1991, Biochem J. 276: 269-270, the entire contentsof which are incorporated herein by reference).

For example, in one embodiment, the beta-lactamase may be a class Aenzyme which includes, but is not limited to, for example, KPC-1, KPC-2,KPC-3 and KPC-4. In another embodiment, the beta-lactamase may be aclass B enzyme which includes, but is not limited to, for example, theIMP family, VIM family, GIM-1 and SPM-1 as well as others. In anotherembodiment, the beta-lactamase may be a class C enzyme such as an AmpCbeta-lactamase. AmpC beta-lactamases hydrolyze broad andextended-spectrum cephalosporins (i.e., cephamycins andoxyimino-beta-lactams). In a further embodiment, the beta-lactamase maybe a class D enzyme that includes, but is not limited to, for example,OXA-23, OXA-24, OXA-25, OXA-26, OXA-27, OXA-40 and OXA-40 as well asothers. In some embodiments, the beta-lactamase may be anextended-spectrum beta-lactamase (ESBL), which hydrolyzes cephalosporinswith an oxyimino chain. ESBLs include, but are not limited to, TEM, SHV,CTX-M, OXA, PER, VEB, GES, and IBC beta-lactamases. In otherembodiments, the beta-lactamase may be an inhibitor-resistant.β-lactamase, optionally selected from an AmpC-type β-lactamases,Carbapenemase, IMP-type carbapenemases (metallo-β-lactamases), VIMs(Verona integron-encoded metallo-β-lactamases), OXA (oxacillinase) groupof β-lactamases, KPCs (K. pneumonia carbapenemases), CMY (Class C), SME,IMI, NMC, CcrA, and NDM (New Delhi metallo-β-lactamases, e.g. NDM-1)beta-lactamases.

In certain embodiments, the beta-lactamase is P1A, P2A, P3A or SYN-004(synonyms for the same enzyme), or P4A. In an embodiment, thebeta-lactamase is P1A or a derivative thereof. The P1A enzyme is arecombinant form of Bacillus licheniformis 749/C small exobeta-lactamase (see WO 2008/065247) which belongs to class A and isgrouped to subgroup 2a in functional classification. B. licheniformisbeta-lactamase and its P1A derivative are considered as penicillinaseswhich have high hydrolytic capacity to degrade e.g. penicillin,ampicillin, amoxicillin or piperacillin and they are generally inhibitedby active site-directed beta-lactamase inhibitors such as clavulanicacid, sulbactam or tazobactam. In another embodiment, the beta-lactamaseis P2A or a derivative thereof as described, for example, in WO2007/147945, the entire contents of which are incorporated herein byreference. The P2A enzyme belongs to class B and is a metallo-enzymethat requires one or two zinc ions as a cofactor for enzyme activity. Inanother embodiment, the beta-lactamase is P3A or a derivative thereof asdescribed, for example, in WO 2011/148041 and U.S. Provisional PatentApplication Nos. 61/980,844 and 62/046,627, the entire contents of allof which are incorporated herein by reference. In a further embodiment,the beta-lactamase is P4A or a derivative thereof as described, forexample, in U.S. Provisional Patent Application Nos. 61/980,844 and62/046,627, the entire contents of all of which are incorporated hereinby reference.

For example, the beta-lactamase may have the sequence of Bacilluslicheniformis PenP, i.e., P1A (SEQ ID NO: 1) or is derived by one ormore mutations of SEQ ID NO: 1. Provided herein is the 263 amino acidsequence of P1A (after removal of a 31 amino acid signal sequence andthe QASKT (Gln-Ala-Ser-Lys-Thr) pentapeptide at the N-terminus, see SEQID NO: 3). As described herein, mutations may be made to this sequenceto generate beta-lactamase derivatives.

SEQ ID NO: 1 Glu Met Lys Asp Asp Phe Ala Lys Leu Glu GluGln Phe Asp Ala Lys Leu Gly Ile Phe Ala LeuAsp Thr Gly Thr Asn Arg Thr Val Ala Tyr ArgPro Asp Glu Arg Phe Ala Phe Ala Ser Thr IleLys Ala Leu Thr Val Gly Val Leu Leu Gln GlnLys Ser Ile Glu Asp Leu Asn Gin Arg Ile ThrTyr Thr Arg Asp Asp Leu Val Asn Tyr Asn ProIle Thr Glu Lys His Val Asp Thr Gly Met ThrLeu Lys Glu Leu Ala Asp Ala Ser Leu Arg TyrSer Asp Asn Ala Ala Gln Asn Leu Ile Leu LysGln Ile Gly Gly Pro Giu Ser Leu Lys Lys GluLeu Arg Lys Ile Gly Asp Glu Val Thr Asn ProGlu Arg Phe Glu Pro Glu Leu Asn Glu Val AsnPro Gly Glu Thr Gln Asp Thr Ser Thr Ala ArgAla Leu Val Thr Ser Leu Arg Ala Phe Ala LeuGlu Asp Lys Leu Pro Ser Glu Lys Arg Glu LeuLeu Ile Asp Trp Met Lys Arg Asn Thr Thr GlyAsp Ala Leu Ile Arg Ala Gly Val Pro Asp GlyTrp Glu Val Ala Asp Lys Thr Gly Ala Ala SerTyr Gly Thr Arg Asn Asp Ile Ala Ile Ile TrpPro Pro Lys Gly Asp Pro Val Val Leu Ala ValLeu Ser Ser Arg Asp Lys Lys Asp Ala Lys TyrAsp Asp Lys Leu Ile Ala Glu Ala Thr Lys ValVal Met Lys Ala Leu Asn Met Asn Gly Lys.

In some embodiments, SEQ ID NO: 1 may have a Met and/or Thr precedingthe first residue of the sequence. In various embodiments, the Met maybe cleaved. As described herein, mutations may be made to the sequencecomprising the Met and/or Thr preceding the first residue to generatebeta-lactamase derivatives.

Also provided herein is the 299 amino acid sequence of P1A beforeremoval of a 31 amino acid signal sequence and the QASKT(Gln-Ala-Ser-Lys-Thr) pentapeptide at the N-terminus as SEQ ID NO: 3:

SEQ ID NO: 3 Met Ile Gln Lys Arg Lys Arg Thr Val Ser PheArg Leu Val Leu Met Cys Thr Leu Leu Phe ValSer Leu Pro Ile Thr Lys Thr Ser Ala Gln AlaSer Lys Thr Glu Met Lys Asp Asp Phe Ala LysLeu Glu Glu Gln Phe Asp Ala Lys Leu Gly IlePhe Ala Leu Asp Thr Gly Thr Asn Arg Thr ValAla Tyr Arg Pro Asp Glu Arg Phe Ala Phe AlaSer Thr Ile Lys Ala Leu Thr Val Gly Val LeuLeu Gln Gln Lys Ser Ile Glu Asp Leu Asn GlnArg Ile Thr Tyr Thr Arg Asp Asp Leu Val AsnTyr Asn Pro Ile Thr Glu Lys His Val Asp ThrGly Met Thr Leu Lys Glu Leu Ala Asp Ala SerLeu Arg Tyr Ser Asp Asn Ala Ala Gln Asn LeuIle Leu Lys Gln Ile Gly Gly Pro Glu Ser LeuLys Lys Glu Leu Arg Lys Ile Gly Asp Glu ValThr Asn Pro Glu Arg Phe Glu Pro Glu Leu AsnGlu Val Asn Pro Gly Glu Thr Gln Asp Thr SerThr Ala Arg Ala Leu Val Thr Ser Leu Arg AlaPhe Ala Leu Glu Asp Lys Leu Pro Ser Glu LysArg Glu Leu Leu Ile Asp Trp Met Lys Arg AsnThr Thr Gly Asp Ala Leu Ile Arg Ala Gly ValPro Asp Gly Trp Glu Val Ala Asp Lys Thr GlyAla Ala Ser Tyr Gly Thr Arg Asn Asp Ile AlaIle Ile Trp Pro Pro Lys Gly Asp Pro Val ValLeu Ala Val Leu Ser Ser Arg Asp Lys Lys AspAla Lys Tyr Asp Asp Lys Leu Ile Ala Glu AlaThr Lys Val Val Met Lys Ala Leu Asn Met Asn Gly Lys

Further, the beta-lactamase polypeptide may include additional upstreamresidues from the first residue of SEQ ID NO: 1 (see, e.g., JBC 258(18): 11211, 1983, the contents of which are hereby incorporated byreference —including the exo-large and exo-small versions of penP andpenP1). Further, the beta-lactamase polypeptide may also includeadditional downstream residues from the last residue of SEQ ID NO: 1.

The polynucleotide sequence of P1A (after removal of a 31 amino acidsignal sequence and the QAKST pentapeptide at the N-terminus) isprovided as SEQ ID NO: 2. As described herein, mutations may be made tothis sequence to generate the beta-lactamase derivatives (including,taking into account degeneracy of the genetic code).

SEQ ID NO: 2 gagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgatttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtggctgataaaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgatgataaacttattgcagaggcaacaaaggtggtaatgaaagccttaa acatgaacggcaaataa

Also provided is the polynucleotide sequence of P1A before the removalof a 31 amino acid signal sequence and the QASKT pentapeptide at theN-terminus as SEQ ID NO: 4. As described herein, mutations may be madeto this sequence to generate beta-lactamase derivatives (including,taking into account degeneracy of the genetic code).

SEQ ID NO: 4 atgattcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagcgcaagcttccaagacggagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtggctgataaaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgatgataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

In some embodiments, mutagenesis of a beta-lactamase is performed toderive advantageous enzymes to be utilized by methods of the presentinvention (e.g. those that can target broad spectra of antibiotics). Insome embodiments, beta-lactamase derivatives are obtained bysite-directed mutagenesis, random mutagenesis, and/or directed evolutionapproaches. In some embodiments, mutation design is based on, interalia, structural data (e.g. crystal structure data, homolog models,etc.) of the following: P1A crystal structure (Knox and Moews, J. MolBiol., 220, 435-455 (1991)), CTX-M-44 (1BZA (Ibuka et al. Journal ofMolecular Biology Volume 285, Issue 5 2079-2087 (1999), 1IYS (Ibuka etal. Biochemistry, 2003, 42 (36): 10634-43), 1IYO, 1IYP and 1IYQ(Shimamura et al. 2002 J. Biol. Chem. 277:46601-08), Proteus vulgaris K1(1HZO, Nugaka at al. J Mol Biol. 2002 Mar. 15; 317(1):109-17) andProteus penneri HugA (Liassine et al. Antimicrob Agents Chemother. 2002January; 46(1):216-9. 2002), and reviewed in Bonnet, Antimicrob. AgentsChemother 48(1): 1-14 (2004) (for CTM-X), the contents of all of whichare herein incorporated by reference in their entirety). In someembodiments, the present mutations are informed by analysis ofstructural data (e.g. crystal structure data, homolog models, etc.) ofany one of the following beta-lactamases: P1A (see, e.g. U.S. Pat. No.5,607,671, the contents of which are hereby incorporated by reference),P2A (see, e.g., WO 2007/147945, the contents of which are herebyincorporated by reference), P3A (see, e.g., WO 2011/148041, the contentsof which are hereby incorporated by reference), CTX-M-3, CTX-M-4,CTX-M-5, CTX-M-9, CTX-M-10, CTX-M-14, CTX-M-15, CTX-M-16, CTX-M-18,CTX-M-19, CTX-M-25, CTX-M-26, CTX-M-27, CTX-M-32, CTX-M-44, CTX-M-45,and CTX-M-54. Such information is available to one skilled in the art ofknown databases, for example, Swiss-Prot Protein Sequence Data Bank,NCBI, and PDB.

In some embodiments, the beta-lactamase includes one or more (e.g. about1, or about 2, or about 3, or about 4, or about 5, or about 6, or about7, or about 8, or about 9, or about 10, or about 15, or about 20, orabout 30, or about 40, or about 50, or about 60, or about 70, or about80, or about 90, or about 100, or about 110, or about 120, or about 130,or about 140, or about 150) mutations to SEQ ID NO: 1 or SEQ ID NO: 3 ora sequence with at least 30%, 35%, 40%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 99.9% identity to SEQ ID NO: 1 or SEQ ID NO:3 (or about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%identity to SEQ ID NO: 1 or SEQ ID NO: 3).

In various embodiments, one or more amino acid of SEQ ID NO: 1 or SEQ IDNO: 3 is substituted with a naturally occurring amino acid, such as ahydrophilic amino acid (e.g. a polar and positively charged hydrophilicamino acid, such as arginine (R) or lysine (K); a polar and neutral ofcharge hydrophilic amino acid, such as asparagine (N), glutamine (Q),serine (S), threonine (T), proline (P), and cysteine (C), a polar andnegatively charged hydrophilic amino acid, such as aspartate (D) orglutamate (E), or an aromatic, polar and positively charged hydrophilicamino acid, such as histidine (H)) or a hydrophobic amino acid (e.g. ahydrophobic, aliphatic amino acid such as glycine (G), alanine (A),leucine (L), isoleucine (I), methionine (M), or valine (V), ahydrophobic, aromatic amino acid, such as phenylalanine (F), tryptophan(W), or tyrosine (Y) or a non-classical amino acid (e.g. selenocysteine,pyrrolysine, N-formylmethionine β-alanine, GABA and δ-Aminolevulinicacid. 4-Aminobenzoic acid (PABA), D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteicacid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β methylamino acids, C α-methyl amino acids, N α-methyl amino acids, and aminoacid analogs in general). In some embodiments, SEQ ID NO: 1 may have aMet and/or Thr preceding the first residue of the sequence. Theseresidues may be similarly mutated as above.

Illustrative mutations are described in U.S. Provisional PatentApplication Nos. 61/980,844 and 62/046,627, the entire contents of allof which are incorporated herein by reference.

In all of the Class A beta-lactamase mutants, the numbering of residuescorresponds to SEQ ID NO: 1. These residue numbers may be converted toAmbler numbers (Ambler et al., 1991, A standard numbering scheme for theClass A β-lactamases, Biochem. J. 276:269-272, the contents of which arehereby incorporated by reference) through use of any conventionalbioinformatic method, for example by using BLAST (Basic Local AlignmentSearch Tools) or FASTA (FAST-All). For example, residue 244 correspondsto Ambler 276. For example, the following conversions may be used:

Ambler Classi- SEQ ID NO: fication No. 1 Residue F33 F6 I72 I44 Q135Q105 G156 G126 T160 T130 A232 A202 A237 A207 A238 A208 S240 S209 T243T212 R244 R213 S266 S234 D276 D244

Furthermore, percent identity may also be assessed with theseconventional bioinformatic methods.

In one embodiment, the beta-lactamase utilized by methods of theinvention comprises an amino acid sequence having at least 60% (e.g.about 60%, or about 61%, or about 62%, or about 63%, or about 64%, orabout 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99%)sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and one or more ofthe following mutations of Ambler classification: F33X, Q135X, G156X,A232X, A237X, A238X, S240X, T243X, R244X, S266X, and D276X, wherein X isany naturally-occurring amino acid and with the proviso that D276X isnot present in the context of a single mutant. In some embodiments, X isa naturally occurring hydrophilic or hydrophobic amino acid residue or anon-classical amino acid.

In another embodiment, the beta-lactamase utilized by methods of theinvention comprises an amino acid sequence having at least 60% sequenceidentity with SEQ ID NO: 1 or SEQ ID NO: 3 and one or more of thefollowing mutations of Ambler classification: a hydrophobic residueother than phenylalanine (F) at position 33; a hydrophobic residue otherthan glutamine (Q) at position 135; a hydrophilic residue other thanglycine (G) at position 156; a hydrophobic residue other than alanine(A) at position 232; a hydrophilic residue other than alanine (A) atposition 237; a hydrophobic or hydrophilic residue other than alanine(A) at position 238; a hydrophilic residue other than serine (S) atposition 240; a hydrophobic residue other than threonine (T) at position243; a hydrophobic residue other than arginine (R) at position 244; ahydrophilic residue other than serine (S) at position 266; and ahydrophilic residue other than aspartate (D) at position 276, with theproviso that hydrophilic amino acid residue other than aspartic acid (D)at a position corresponding to position 276 is not present in thecontext of a single mutant.

As used throughout, a hydrophilic amino acid residue may include a polarand positively charged hydrophilic residue selected from arginine (R)and lysine (K), a polar and neutral of charge hydrophilic residueselected from asparagine (N), glutamine (Q), serine (S), threonine (T),proline (P), and cysteine (C), a polar and negatively chargedhydrophilic residue selected from aspartate (D) and glutamate (E), or anaromatic, polar and positively charged hydrophilic including histidine(H). As used throughout, a hydrophobic amino acid residue may include ahydrophobic, aliphatic amino acid selected from glycine (G), alanine(A), leucine (L), isoleucine (I), methionine (M), or valine (V) or ahydrophobic, aromatic amino acid selected from phenylalanine (F),tryptophan (W), or tyrosine (Y).

Mutations may be made to the gene sequence of a beta-lactamase (e.g. SEQID NOs: 3 and 4) by reference to the genetic code, including taking intoaccount codon degeneracy.

In some embodiments, the beta-lactamase utilized by methods of theinvention comprises one or more of the following mutations at positionsof Ambler classification: F33Y, Q135M, G156R, A232G, A237S, A238G or T,S240P or D, T2431, R244T, S266N, D276N or R or K, provided that D276N orR or K is not in the context of a single mutant. In one embodiment, thebeta-lactamases comprise Q135M. In another embodiment, thebeta-lactamases comprise G156R and A238T. In another embodiment, thebeta-lactamases comprise F33Y and D276N. In still another embodiment,the beta-lactamases comprise F33Y, S240P, and D276N. In one embodiment,the beta-lactamases comprise F33Y, A238T, and D276N. In anotherembodiment, the beta-lactamases comprise A232G, A237S, A238G, and S240D.In a further embodiment, the beta-lactamases comprise A232G, A237S,A238G, S240D, and R244T. In another embodiment, the beta-lactamasescomprise A232G, A237S, A238G, S240D, and D276R. In one embodiment, thebeta-lactamases comprise A232G, A237S, A238G, S240D, and D276K. In oneembodiment, the beta-lactamases comprise A232G, A237S, A238G, S240D, andQ135M. In one embodiment, the beta-lactamases comprise A238T. In oneembodiment, the beta-lactamases comprise T2431, S266N, and D276N. In oneembodiment, the beta-lactamases comprise A232G, A237S, A238G, S240D, andD276N.

In other embodiments, the beta-lactamases comprise an amino acidsequence having at least 60% sequence identity with SEQ ID NO: 1 or SEQID NO: 3 and the following of Ambler classification: a hydrophobicresidue other than alanine (A) at position 232; a hydrophilic residueother than alanine (A) at position 237; a hydrophobic residue other thanalanine (A) at position 238; a hydrophilic residue other than serine (S)at position 240; and a hydrophilic residue other than aspartate (D) atposition 276. In some embodiments, the hydrophobic residue other thanalanine (A) at position 232 is glycine (G). In some embodiments, thehydrophilic residue other than alanine (A) at position 237 is serine(S). In some embodiments, the hydrophobic residue other than alanine (A)at position 238 is glycine (G). In some embodiments, the hydrophilicresidue other than serine (S) at position 240 is aspartate (D). In someembodiments, the other than aspartate (D) at position 276 is asparagine(N). In some embodiments, the beta-lactamase and/or pharmaceuticalcomposition comprises one or more of A232G, A237S, A238G, S240D, andD276N. In some embodiments, the beta-lactamase comprises all of A232G,A237S, A238G, S240D, and D276N, the sequence of which is SEQ ID NO:5:

SEQ ID NO: 5 EMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVGDKTGSGDYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAKYDNKLIAEATKVVMMLNM NGK or

SEQ ID NO: 6 is derived from SEQ ID NO: 5, and further includes thesignal and the addition of the QASKT amino acids:

SEQ ID NO: 6 MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAQASKTEMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITYTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVGDKTGSGDYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAKYDNKLIAEATKVVMKALNMNGK

An illustrative polynucleotide of the invention is SEQ ID NO: 7:

SEQ ID NO: 7 Atgttcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagcgcaagcttccaagacggagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtgggtgataaaactggaagcggagattatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgataataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

Full nucleotide sequence of A232G, A237S, A238G, S240D, and D276Nmutant, Hind III site (AAGCTT-in bold) and additional K and T aminoacids. The leader and additional nucleotides (Hind III site and K and Tamino acids—for the addition of the amino acid sequence QASKT) areunderlined.

Additional sequences of beta-lactamases including P1A, P2A, P3A, and P4Aand derivatives thereof are described for example, in WO2011/148041 andU.S. Provisional Patent Application Nos. 61/980,844 and 62/046,627, theentire contents of all of which are incorporated herein by reference.The following table lists representative beta-lactamases and theirderivatives which can be utilized in methods of the present invention:

Mutations relative to P1A (based on the Ambler classification) Name Wildtype RS310 (or P1A) D276N IS118 (or P3A) I72S IS222 T160F IS203 R244TIS217 R244T D276K IS215 Q135M IS197 G156R A238T IS235 F33Y D276N IS158F33Y S240P D276N IS230 (or IS181) F33Y A238T D276N IS232 (or IS180) I72SQ135M T160F JS227 (Block 1 mutants) A232G A237S A238G S240D IS191 (Block2 mutants) A232G A237S A238G S240D R244T IS229 A232G A237S A238G S240DD276R IS219 A232G A237S A238G S240D D276K IS221 A232G A237S A238G S240DQ135M IS224 A238T IS233 T243I S266N D276N IS234 (or IS176) A232G A237SA238G S240D D276N IS288 (or P4A)

In various embodiments, the beta-lactamases possess desirablecharacteristics, including, for example, having an ability toefficiently target a broad spectra of antibiotics including oralantibiotics. In various embodiments, the beta-lactamases possessdesirable enzyme kinetic characteristics. For example, in someembodiments, the beta-lactamases possess a low K_(M) for at least oneoral antibiotic, including, for example, a K_(M) of less than about 500μM, or about 100 μM, or about 10 μM, or about 1 μM, or about 0.1 μM (100nM), or about 0.01 μM (10 nM), or about 1 nM. In various embodiments,the beta-lactamases possess a high V_(max) for at least one oralantibiotic, including, for example, V_(max) which is greater than about100 s-1, or about 1000 s-1, or about 10000 s-1, or about 100000 s-1, orabout 1000000 s-1. In various embodiments, the beta-lactamases possesscatalytic efficiency that is greater than about 106 M⁻¹ s⁻¹ for at leastone oral antibiotic.

In various embodiments, the beta-lactamases are stable and/or active inthe GI tract, e.g. in one or more of the mouth, esophagus, stomach,duodenum, small intestine, duodenum, jejunum, ileum, large intestine,colon transversum, colon descendens, colon ascendens, colon sigmoidenum,cecum, and rectum. In a specific embodiment, the beta-lactamase isstable in the large intestine, optionally selected from one or more ofcolon transversum, colon descendens, colon ascendens, colon sigmoidenumand cecum. In a specific embodiment, the beta-lactamase is stable in thesmall intestine, optionally selected from one or more of duodenum,jejunum, and ileum. In some embodiments, the beta-lactamase is resistantto proteases in the GI tract, including for example, the smallintestine. In some embodiments, the beta-lactamase is substantiallyactive at a pH of about 6.0 to about 7.5, e.g. about 6.0, or about 6.1,or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, orabout 6.7, or about 6.8, or about 6.9, or about 7.0, or about 7.1, orabout 7.2, or about 7.3, or about 7.4, or about 7.5 (including, forexample, via formulation, as described herein). In various embodiments,the beta-lactamases of the present invention are resistant to one ormore beta-lactamase inhibitors, optionally selected from avibactam,tazobactam, sulbactam, and clavulanic acid. In other embodiments, asdescribed herein the beta-lactamases of the present invention aresusceptible to one or more beta-lactamase inhibitors and this propertyis exploited to ensure antibiotic hydrolysis does not interfere with thetherapeutic benefit of the oral antibiotic. In some embodiments, stablerefers to an enzyme that has a long enough half-life and maintainssufficient activity for therapeutic effectiveness.

In some embodiments, the beta-lactamases described herein, includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the beta-lactamase such that covalent attachmentdoes not prevent the activity of the enzyme. For example, but not by wayof limitation, derivatives include beta-lactamases that have beenmodified by, inter alia, glycosylation, lipidation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications canbe carried out, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative can contain one or more non-classicalamino acids.

In still other embodiments, the beta-lactamases described herein may bemodified to add effector moieties such as chemical linkers, detectablemoieties such as for example fluorescent dyes, enzymes, substrates,bioluminescent materials, radioactive materials, and chemiluminescentmoieties, or functional moieties such as for example streptavidin,avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactivematerials.

The beta-lactamases described herein can possess a sufficiently basicfunctional group, which can react with an inorganic or organic acid, ora carboxyl group, which can react with an inorganic or organic base, toform a pharmaceutically acceptable salt. A pharmaceutically acceptableacid addition salt is formed from a pharmaceutically acceptable acid, asis well known in the art. Such salts include the pharmaceuticallyacceptable salts listed in, for example, Journal of PharmaceuticalScience, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts;Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.),Verlag, Zurich (Switzerland) 2002, which are hereby incorporated byreference in their entirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, o-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thebeta-lactamases having an acidic functional group, such as a carboxylicacid functional group, and a base. Suitable bases include, but are notlimited to, hydroxides of alkali metals such as sodium, potassium, andlithium; hydroxides of alkaline earth metal such as calcium andmagnesium; hydroxides of other metals, such as aluminum and zinc;ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, the compositions described herein are in the formof a pharmaceutically acceptable salt.

Further, any beta-lactamases described herein can be administered to asubject as a component of a composition that comprises apharmaceutically acceptable carrier or vehicle. Such compositions canoptionally comprise a suitable amount of a pharmaceutically acceptableexcipient so as to provide the form for proper administration.

Pharmaceutical excipients can be liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical excipients can be, for example, saline, gum acacia,gelatin, starch paste, talc, keratin, colloidal silica, urea and thelike. In addition, auxiliary, stabilizing, thickening, lubricating, andcoloring agents can be used. In one embodiment, the pharmaceuticallyacceptable excipients are sterile when administered to a subject. Wateris a useful excipient when any agent described herein is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid excipients, specifically forinjectable solutions. Suitable pharmaceutical excipients also includestarch, glucose, cellulose, hypromellose, lactose, sucrose, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene, glycol, povidone,crosspovidone, water, ethanol and the like. Any agent described herein,if desired, can also comprise minor amounts of wetting or emulsifyingagents, or pH buffering agents. Other examples of suitablepharmaceutical excipients are described in Remington's PharmaceuticalSciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995),incorporated herein by reference.

Where necessary, the beta-lactamases and/or pharmaceutical compositions(and/or additional therapeutic agents) can include a solubilizing agent.Also, the agents can be delivered with a suitable vehicle or deliverydevice. Compositions for administration can optionally include a localanesthetic such as, for example, lignocaine to lessen pain at the siteof the injection. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.

Oral Antibiotics

In various embodiments, the beta-lactamases deactivate one or more oralantibiotics. In various embodiments, the beta-lactamases hydrolyze oneor more oral antibiotics. In various embodiments, the describedbeta-lactamases and/or pharmaceutical compositions (and/or additionaltherapeutic agents) are formulated in a manner that preserves thetherapeutic (e.g. systemic) action of one or more oral antibiotics whilepreventing the action of excess amounts these oral antibiotics lower inthe GI tract, where they may disrupt the GI microbiota.

For example, the described beta-lactamases and/or pharmaceuticalcompositions (and/or additional therapeutic agents) are formulated in amanner that active antibiotic that is not absorbed from the GI tractafter an oral dose or is returned in active form to the intestinal tractfrom the systemic circulation is deactivated. In certain embodiments,the orally administered antibiotics are selected from penicillins,cephalosporins, monobactams, and carbapenems.

Penicillins include, for example, Amdinocillin, Amoxicillin (e.g.NOVAMOX, AMOXIL); Ampicillin (e.g. PRINCIPEN); Azlocillin; Carbenicillin(e.g. GEOCILLIN); Cloxacillin (e.g. TEGOPEN); Cyclacillin, Dicloxacillin(e.g. DYNAPEN); Flucloxacillin (e.g. FLOXAPEN); Mezlocillin (e.g.MEZLIN); Methicillin (e.g. STAPHCILLIN); Nafcillin (e.g. UNIPEN);Oxacillin (e.g. PROSTAPHLIN); Penicillanic Acid, Penicillin G (e.g.PENTIDS or PFIZERPEN); Penicillin V (e.g. VEETIDS (PEN-VEE-K));Piperacillin (e.g. PIPRACIL); Sulbactam, Temocillin (e.g. NEGABAN); andTicarcillin (e.g. TICAR).

Illustrative Penicillins Include:

Generic Brand Name Amoxicillin AMOXIL, POLYMOX, TRIMOX, WYMOX AmpicillinOMNIPEN, POLYCILLIN, POLYCILLIN-N, PRINCIPEN, TOTACILLIN BacampicillinSPECTROBID Carbenicillin GEOCILLIN, GEOPEN Cloxacillin CLOXAPENDicloxacillin DYNAPEN, DYCILL, PATHOCIL Flucloxacillin FLOPEN, FLOXAPEN,STAPHCILLIN Mezlocillin MEZLIN Nafcillin NAFCIL, NALLPEN, UNIPENOxacillin BACTOCILL, PROSTAPHLIN Penicillin G BICILLIN L-A, CRYSTICILLIN300 A.S., PENTIDS, PERMAPEN, PFIZERPEN, PFIZERPEN-AS, WYCILLINPenicillin V BEEPEN-VK, BETAPEN-VK, LEDERCILLIN VK, V-CILLIN KPiperacillin PIPRACIL Pivampicillin Pivmecillinam Ticarcillin TICAR

Cephalosporins include, for example, a first generation cephalosporin(e.g. Cefadroxil (e.g. DURICEF); Cefazolin (e.g. ANCEF); Ceftolozane,Cefalotin/Cefalothin (e.g. KEFLIN); Cefalexin (e.g. KEFLEX); a secondgeneration cephalosporin (e.g. Cefaclor (e.g. DISTACLOR); Cefamandole(e.g. MANDOL); Cefoxitin (e.g. MEFOXIN); Cefprozil (e.g. CEFZIL);Cefuroxime (e.g. CEFTIN, ZINNAT)); a third generation cephalosporin(e.g. Cefixime (e.g. SUPRAX); Cefdinir (e.g. OMNICEF, CEFDIEL);Cefditoren (e.g. SPECTRACEF); Cefoperazone (e.g. CEFOBID); Cefotaxime(e.g. CLAFORAN); Cefpodoxime (e.g. VANTIN); Ceftazidime (e.g. FORTAZ);Ceftibuten (e.g. CEDAX) Ceftizoxime (e.g. CEFIZOX); and Ceftriaxone(e.g. ROCEPHIN)); a fourth generation cephalosporin (e.g. Cefepime (e.g.MAXIPIME)); or a fifth generation cephalosporin (e.g. Ceftarolinefosamil (e.g. TEFLARO); Ceftobiprole (e.g. ZEFTERA)). Also included isLatamoxef (or moxalactam). In a specific embodiment, cephalosporinsinclude, for example, cefoperazone, ceftriaxone or cefazolin.

Generic Brand Name First Generation Cefacetrile (cephacetrile) CELOSPOR,CELTOL, CRISTACEF Cefadroxil (cefadroxyl) DURICEF, ULTRACEF Cefalexin(cephalexin) KEFLEX, KEFTAB Cefaloglycin (cephaloglycin) KEFGLYCINCefalonium (cephalonium) Cefaloridine (cephaloradine) Cefalotin(cephalothin) KEFLIN Cefapirin (cephapirin) CEFADYL CefatrizineCefazaflur Cefazedone Cefazolin (cephazolin) ANCEF, KEFZOL Cefradine(cephradine) VELOSEF Cefroxadine Ceftezole Second Generation CefaclorCECLOR, CECLOR CD, DISTACLOR, KEFLOR, RANICOR Cefamandole MANDOLCefmetazole Cefonicid MONOCID Cefotetan CEFOTAN Cefoxitin MEFOXINCefprozil (cefproxil) CEFZIL Cefuroxime CEFTIN, KEFUROX, ZINACEF, ZINNATCefuzonam Third Generation Cefcapene Cefdaloxime Cefdinir OMNICEF,CEFDIEL Cefditoren SPECTRACEF Cefetamet Cefixime SUPRAX CefmenoximeCEFMAX Cefodizime Cefotaxime CLAFORAN Cefpimizole Cefpodoxime VANTINCefteram Ceftibuten CEDAX Ceftiofur EXCEDE Ceftiolene CeftizoximeCEFIZOX Ceftriaxone ROCEPHIN Cefoperazone CEFOBID Ceftazidime CEPTAZ,FORTUM, FORTAZ, TAZICEF, TAZIDIME Fourth Generation Cefclidine CefepimeMAXIPIME Cefluprenam Cefoselis Cefozopran Cefpirome CEFROM CefquinomeFifth Generation Ceftobiprole ZEFTERA Ceftaroline TEFLARO Not ClassifiedCefaclomezine Cefaloram Cefaparole Cefcanel Cefedrolor CefempidoneCefetrizole Cefivitril Cefmatilen Cefmepidium Cefovecin CefoxazoleCefrotil Cefsumide Cefuracetime Ceftioxide

Monobactams include, for example, aztreonam (e.g. AZACTAM, CAYSTON),tigemonam, nocardicin A, and tabtoxin.

Carbapenems include, for example, meropenem, imipenem (by way ofnon-limiting example, imipenem/cilastatin), ertapenem, doripenem,panipenem/betamipron, biapenem, razupenem (PZ-601), tebipenem,lenapenem, and tomopenem. Carbapenems also include thienamycins.

Illustrative Carbapenems Include

Generic Brand Name Imipenem, PRIMAXIN Imipenem/cilastatin DoripenemDORIBAX Meropenem MERREM Ertapenem INVANZ

Beta-Lactamase Inhibitors

In various embodiments, the described beta-lactamases and/orpharmaceutical compositions (and/or additional therapeutic agents) areformulated in a manner that preserves the therapeutic (e.g. systemic)action of one or more oral antibiotics while preventing the action ofresidual or excess amounts these oral antibiotics (e.g. activeantibiotic that is not absorbed from the GI tract after an oral dose oris returned in active form to the intestinal tract from the systemiccirculation) lower in the GI tract, where they may disrupt the GImicrobiota and this dual purpose is effected, in part, by use of one ormore beta-lactamase inhibitor.

For example, the described beta-lactamases may be administered in apatient that receives one or more beta-lactamase inhibitors (e.g.sequential or simultaneous co-administration, or co-formulation) suchthat the one or more beta-lactamase inhibitors act to protect the oralantibiotics higher in the GI tract (e.g. ileum and above, or theproximal small intestine) by reducing or eliminating beta-lactamaseactivity. However, the one or more beta-lactamase inhibitors do not havesuch inhibitory effects on beta-lactamase activity lower in the GI tract(e.g. distal small intestine and/or the colon) and therefore allow thedescribed beta-lactamase to deactivate (e.g. hydrolyze) residual orexcess oral antibiotic lower in the GI tract and thus prevent ormitigate damage to the GI microbiota.

In some embodiments, the beta-lactamase inhibitor tracks with thebeta-lactam antibiotic such that both are available for absorption inthe proximal small intestine. The beta-lactamase inhibitor serves toprotect the beta-lactam antibiotic from the beta-lactamase in theproximal small intestine. The antibiotic and the inhibitor are then bothabsorbed into the bloodstream and thereby removed from the proximalsmall intestine. As the concentration of inhibitor decreases in thesmall intestine, the beta-lactamase becomes active. Any residual orexcess antibiotic that remains in the intestine or re-enters with thebile will is inactivated prior to encountering the colonic microbiome.

In some embodiments, the beta-lactamase inhibitor includes, for example,tazobactam, sulbactam, clavulanic acid, avibactam, monobactamderivatives, ATMO derivatives, penems (e.g., BRL42715 and derivativesthereof, Syn1012, oxapenems, trinems, 1-β-methylcarbapenems), penicillinand cephalosporin sulfone derivatives (e.g., C-2/C-3-substitutedpenicillin and cephalosporin sulfones, C-6-substituted penicillinsulfones), non-β-lactam inhibitors (e.g., boronic acid transition stateanalogs, phophonates, NXL104, hydroxmates) and metallo-β-lactamaseinhibitors such as thiol derivatives, pyridine dicarboxylates,trifluoromethyl ketones and alcohols, carbapenem analogs, tricyclicnatural products, succinate derivatives, and C-6-mercaptomethylpenicillinates. Co-formulations of an oral antibiotic with one or morebeta-lactamase inhibitors are also provided in some embodiments (e.g.Augmentin is a mixture of amoxicillin and clavulanic acid; Sultamicillinis a mixture of ampicillin and sulbactam).

Further, any of the beta-lactamase inhibitors described in Drawz, ClinMicrobiol Rev. January 2010; 23(1): 160-201, the contents of which arehereby incorporated by reference in their entirety, are encompassed bythe present invention.

Formulations and Administration

The present invention includes the described beta-lactamases and/orpharmaceutical compositions (and/or additional therapeutic agents) invarious formulations. Any beta-lactamase and/or pharmaceuticalcomposition (and/or additional therapeutic agents) described herein cantake the form of solutions, suspensions, emulsion, drops, tablets,pills, pellets, capsules, capsules containing liquids, capsulescontaining multiparticulates, powders, suppositories, emulsions,aerosols, sprays, suspensions, delayed-release formulations,sustained-release formulations, controlled-release formulations, or anyother form suitable for use. In one embodiment, the composition is inthe form of a capsule or a tablet (see, e.g., U.S. Pat. No. 5,698,155).

The formulations comprising the beta-lactamases and/or pharmaceuticalcompositions (and/or additional therapeutic agents) may conveniently bepresented in unit dosage forms. For example, the dosage forms may beprepared by methods which include the step of bringing the therapeuticagents into association with a carrier, which constitutes one or moreaccessory ingredients. Typically, the formulations are prepared byuniformly and intimately bringing the therapeutic agent into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product into dosage forms of the desiredformulation (e.g., wet or dry granulation, powder blends, etc., followedby press tableting)

In one embodiment, the beta-lactamases (and/or additional therapeuticagents) described herein is formulated as a composition adapted for amode of administration described herein.

In some embodiments, the administration the beta-lactamases and/orpharmaceutical compositions (and/or additional therapeutic agents) isany one of oral, intravenous, and parenteral. In some embodiments, theadministration of the beta-lactamases and/or pharmaceutical compositions(and/or additional therapeutic agents) is not intravenous in order to,for example, prevent interference with an antibiotic administeredsystemically. In other embodiments, routes of administration include,for example: oral, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal,intracerebral, intravaginal, transdermal, rectally, by inhalation, ortopically, particularly to the ears, nose, eyes, or skin. In someembodiments, the administering is effected orally or by parenteralinjection.

In various embodiments, the administration the beta-lactamases and/orpharmaceutical compositions (and/or additional therapeutic agents) isinto the GI tract via, for example, oral delivery, nasogastral tube,intestinal intubation (e.g. an enteral tube or feeding tube such as, forexample, a jejunal tube or gastro-jejunal tube, etc.), endoscopy,colonoscopy, or enema.

In an embodiment, the beta-lactamases and/or pharmaceutical compositions(and/or additional therapeutic agents) described herein can beadministered orally. In other embodiments, the beta-lactamases and/orpharmaceutical compositions (and/or additional therapeutic agents) canalso be administered by any other convenient route, for example, byintravenous infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and can be administered together with anadditional therapeutic agent. Administration can be systemic or local.In some embodiments, administration is not at the site of infection toavoid, for example, hydrolysis of an antibiotic at the site ofinfection. Various delivery systems are known, e.g., encapsulation inliposomes, microparticles, microcapsules, capsules, etc., and can beused for administration.

In specific embodiments, it may be desirable to administer locally tothe area in need of treatment.

In one embodiment, the beta-lactamases and/or pharmaceuticalcompositions (and/or additional therapeutic agents) described herein isformulated as a composition adapted for oral administration to humans.Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, sprinkles, emulsions,capsules, syrups, or elixirs, for example. Orally administeredcompositions can comprise one or more agents, for example, sweeteningagents such as fructose, aspartame or saccharin; flavoring agents suchas peppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration to provide a sustained action over an extendedperiod of time. Selectively permeable membranes surrounding anosmotically active agent driving any beta-lactamases (and/or additionaltherapeutic agents) described herein are also suitable for orallyadministered compositions. In these latter platforms, fluid from theenvironment surrounding the capsule is imbibed by the driving compound,which swells to displace the agent or agent composition through anaperture. These delivery platforms can provide an essentially zero orderdelivery profile as opposed to the spiked profiles of immediate releaseformulations. A time-delay material such as glycerol monostearate orglycerol stearate can also be useful. Oral compositions can includestandard excipients such as mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose, ethacrylic acid and derivativepolymers thereof, and magnesium carbonate. In one embodiment, theexcipients are of pharmaceutical grade. Suspensions, in addition to theactive compounds, may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, tragacanth, etc., and mixtures thereof.

In various embodiments, the beta-lactamases and/or pharmaceuticalcompositions (and/or additional therapeutic agent) are formulated assolid dosage forms such as tablets, dispersible powders, granules, andcapsules. In one embodiment, the beta-lactamases and/or pharmaceuticalcompositions (and/or additional therapeutic agent) are formulated as acapsule. In another embodiment, the beta-lactamases and/orpharmaceutical compositions (and/or additional therapeutic agent) areformulated as a tablet. In yet another embodiment, the beta-lactamasesand/or pharmaceutical compositions (and/or additional therapeutic agent)are formulated as a soft-gel capsule. In a further embodiment, thebeta-lactamases and/or pharmaceutical compositions (and/or additionaltherapeutic agent) are formulated as a gelatin capsule.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents.

Various methods may be used to formulate and/or deliver the agentsdescribed herein to a location of interest. For example, thebeta-lactamases and/or pharmaceutical compositions (and/or additionaltherapeutic agents) described herein may be formulated for delivery tothe gastrointestinal tract. The gastrointestinal tract includes organsof the digestive system such as mouth, esophagus, stomach, duodenum,small intestine, large intestine and rectum and includes all subsectionsthereof (e.g. the small intestine may include the duodenum, jejunum andileum; the large intestine may include the colon transversum, colondescendens, colon ascendens, colon sigmoidenum and cecum). For example,the beta-lactamases and/or pharmaceutical compositions (and/oradditional therapeutic agents) described herein may be formulated fordelivery to one or more of the stomach, small intestine, large intestineand rectum and includes all subsections thereof (e.g. duodenum, jejunumand ileum, colon transversum, colon descendens, colon ascendens, colonsigmoidenum and cecum). In some embodiments, the compositions describedherein may be formulated to deliver to the upper or lower GI tract. Inan embodiment, the beta-lactamases and/or pharmaceutical compositions(and/or additional therapeutic agents) may be administered to a subject,by, for example, directly or indirectly contacting the mucosal tissuesof the gastrointestinal tract.

For example, in various embodiments, the present invention providesmodified release formulations comprising at least one beta-lactamase(and/or additional therapeutic agents), wherein the formulation releasesa substantial amount of the beta-lactamase (and/or additionaltherapeutic agents) into one or more regions of the GI tract. Forexample, the formulation may release at least about 60% of thebeta-lactamase after the stomach and into one or more regions of the GItract. In various embodiments, the modified release formulationscomprising at least one beta-lactamase (and/or additional therapeuticagents) are released in a manner that allows for the therapeutic (e.g.systemic) activity of one or more oral antibiotic (and/or abeta-lactamase inhibitor) but prevents or mitigates the deleteriouseffects of residual or excess oral antibiotics on the microbiota of theGI tract (e.g. active antibiotic that is not absorbed from the GI tractafter an oral dose or is returned in active form to the intestinal tractfrom the systemic circulation). In various embodiments, the modifiedrelease formulations comprising at least one beta-lactamase (and/oradditional therapeutic agents) are released distal to the release of oneor more oral antibiotic (and/or a beta-lactamase inhibitor). Forexample, in various embodiments, the modified release formulationscomprising at least one beta-lactamase (and/or additional therapeuticagents) are released distal to the ileum and below. For example, invarious embodiments, the modified release formulations comprising atleast one beta-lactamase (and/or additional therapeutic agents) arereleased is released in the distal small intestine and/or the colon.

In various embodiments, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) after the stomach into one or more regions of theintestine. For example, the modified-release formulation releases atleast 60%, at least 61%, at least 62%, at least 63%, at least 64%, atleast 65%, at least 66%, at least 67%, at least 68%, at least 69%, atleast 70%, at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% of the beta-lactamase (or additional therapeutic agents) in theintestine.

In various embodiments, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the small intestine. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the beta-lactamase(oradditional therapeutic agents) in the small intestine.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the duodenum. For example, the modified-releaseformulation releases at least 60%, at least 61%, at least 62%, at least63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% of the beta-lactamase (or additionaltherapeutic agents) in the duodenum.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the jejunum. For example, the modified-releaseformulation releases at least 60%, at least 61%, at least 62%, at least63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% of the beta-lactamase (or additionaltherapeutic agents) in the jejunum.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the ileum and/or the ileocecal junction. Forexample, the modified-release formulation releases at least 60%, atleast 61%, at least 62%, at least 63%, at least 64%, at least 65%, atleast 66%, at least 67%, at least 68%, at least 69%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% of thebeta-lactamase (or additional therapeutic agents) in the ileum and/orthe ileocecal junction.

In various embodiments, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the large intestine. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the beta-lactamase (oradditional therapeutic agents) in the large intestine.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the cecum. For example, the modified-releaseformulation releases at least 60%, at least 61%, at least 62%, at least63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% of the beta-lactamase (or additionaltherapeutic agents) in the cecum.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the ascending colon. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the beta-lactamase (oradditional therapeutic agents) in the ascending colon.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the transverse colon. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the beta-lactamase (oradditional therapeutic agents) in the transverse colon.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the descending colon. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the beta-lactamase (oradditional therapeutic agents) in the descending colon.

In one embodiment, the modified-release formulation of the presentinvention releases at least 60% of the beta-lactamase (or additionaltherapeutic agents) in the sigmoid colon. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the beta-lactamase (oradditional therapeutic agents) in the sigmoid colon.

In various embodiments, the modified-release formulation does notsubstantially release the beta-lactamase (or additional therapeuticagents) in the stomach.

In certain embodiments, the modified-release formulation releases thebeta-lactamase (or additional therapeutic agents) at a specific pH. Forexample, in some embodiments, the modified-release formulation issubstantially stable in an acidic environment and substantially unstable(e.g., dissolves rapidly or is physically unstable) in a near neutral toalkaline environment. In some embodiments, stability is indicative ofnot substantially releasing while instability is indicative ofsubstantially releasing. For example, in some embodiments, themodified-release formulation is substantially stable at a pH of about7.0 or less, or about 6.7 or less, or about 6.5 or less, or about 6.2 orless or about 6.0 or less, or about 5.5 or less, or about 5.0 or less,or about 4.5 or less, or about 4.0 or less, or about 3.5 or less, orabout 3.0 or less, or about 2.5 or less, or about 2.0 or less, or about1.5 or less, or about 1.0 or less. In some embodiments, the presentformulations are stable in lower pH areas and therefore do notsubstantially release in, for example, the stomach. In some embodiments,modified-release formulation is substantially stable at a pH of about 1to about 4 or lower and substantially unstable at pH values that aregreater. In these embodiments, the modified-release formulation is notsubstantially released in the stomach. In these embodiments, themodified-release formulation is substantially released in the smallintestine (e.g. one or more of the duodenum, jejunum, and ileum) and/orlarge intestine (e.g. one or more of the cecum, ascending colon,transverse colon, descending colon, and sigmoid colon). In someembodiments, modified-release formulation is substantially stable at apH of about 4 to about 5 or lower and consequentially is substantiallyunstable at pH values that are greater and therefore is notsubstantially released in the stomach and/or small intestine (e.g. oneor more of the duodenum, jejunum, and ileum). In these embodiments, themodified-release formulation is substantially released in the largeintestine (e.g. one or more of the cecum, ascending colon, transversecolon, descending colon, and sigmoid colon). In an embodiment, themodified-release formulation is substantially unstable at a pH ofgreater than about 6.2. In another embodiment, the modified-releaseformulation is substantially unstable at a pH of greater than about 6.7.In various embodiments, the pH values recited herein may be adjusted asknown in the art to account for the state of the subject, e.g. whetherin a fasting or postprandial state.

In some embodiments, the modified-release formulation is substantiallystable in gastric fluid and substantially unstable in intestinal fluidand, accordingly, is substantially released in the small intestine (e.g.one or more of the duodenum, jejunum, and ileum) and/or large intestine(e.g. one or more of the cecum, ascending colon, transverse colon,descending colon, and sigmoid colon).

In some embodiments, the modified-release formulation is stable ingastric fluid or stable in acidic environments. These modified-releaseformulations release about 30% or less by weight of the beta-lactamaseand/or additional therapeutic agent in the modified-release formulationin gastric fluid with a pH of about 4 to about 5 or less, or simulatedgastric fluid with a pH of about 4 to about 5 or less, in about 15, orabout 30, or about 45, or about 60, or about 90 minutes.Modified-release formulations of the of the invention may release fromabout 0% to about 30%, from about 0% to about 25%, from about 0% toabout 20%, from about 0% to about 15%, from about 0% to about 10%, about5% to about 30%, from about 5% to about 25%, from about 5% to about 20%,from about 5% to about 15%, from about 5% to about 10% by weight of thebeta-lactamase and/or additional therapeutic agent in themodified-release formulation in gastric fluid with a pH of 4-5, or lessor simulated gastric fluid with a pH of 4-5 or less, in about 15, orabout 30, or about 45, or about 60, or about 90 minutes.Modified-release formulations of the invention may release about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, or about 10% by weight of the total beta-lactamase and/oradditional therapeutic agent in the modified-release formulation ingastric fluid with a pH of 5 or less, or simulated gastric fluid with apH of 5 or less, in about 15, or about 30, or about 45, or about 60, orabout 90 minutes.

In some embodiments, the modified-release formulation is unstable inintestinal fluid. These modified-release formulations release about 70%or more by weight of the beta-lactamase and/or additional therapeuticagent in the modified-release formulation in intestinal fluid orsimulated intestinal fluid in about 15, or about 30, or about 45, orabout 60, or about 90 minutes. In some embodiments, the modified-releaseformulation is unstable in near neutral to alkaline environments. Thesemodified-release formulations release about 70% or more by weight of thebeta-lactamase and/or additional therapeutic agent in themodified-release formulation in intestinal fluid with a pH of about 4-5or greater, or simulated intestinal fluid with a pH of about 4-5 orgreater, in about 15, or about 30, or about 45, or about 60, or about 90minutes. A modified-release formulation that is unstable in near neutralor alkaline environments may release 70% or more by weight ofbeta-lactamase and/or additional therapeutic agent in themodified-release formulation in a fluid having a pH greater than about 5(e.g., a fluid having a pH of from about 5 to about 14, from about 6 toabout 14, from about 7 to about 14, from about 8 to about 14, from about9 to about 14, from about 10 to about 14, or from about 11 to about 14)in from about 5 minutes to about 90 minutes, or from about 10 minutes toabout 90 minutes, or from about 15 minutes to about 90 minutes, or fromabout 20 minutes to about 90 minutes, or from about 25 minutes to about90 minutes, or from about 30 minutes to about 90 minutes, or from about5 minutes to about 60 minutes, or from about 10 minutes to about 60minutes, or from about 15 minutes to about 60 minutes, or from about 20minutes to about 60 minutes, or from about 25 minutes to about 90minutes, or from about 30 minutes to about 60 minutes.

Examples of simulated gastric fluid and simulated intestinal fluidinclude, but are not limited to, those disclosed in the 2005Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or othersimulated gastric fluids and simulated intestinal fluids known to thoseof skill in the art, for example, simulated gastric fluid and/orintestinal fluid prepared without enzymes.

In various embodiments, the modified-release formulations comprising abeta-lactamase are substantially stable in chyme. For example, there is,in some embodiments, a loss of less than about 50% or about 40%, orabout 30%, or about 20%, or about 10% of beta-lactamase activity inabout 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 hour fromadministration.

In various embodiments, the modified-release formulation of the presentinvention may utilize one or more modified-release coatings such asdelayed-release coatings to provide for effective, delayed yetsubstantial delivery of the beta-lactamase to the GI tract togetherwith, optionally, additional therapeutic agents. In one embodiment, thedelayed-release coating includes an enteric agent that is substantiallystable in acidic environments and substantially unstable in near neutralto alkaline environments. In an embodiment, the delayed-release coatingcontains an enteric agent that is substantially stable in gastric fluid.The enteric agent can be selected from, for example, solutions ordispersions of methacrylic acid copolymers, cellulose acetate phthalate,hydroxypropylmethyl cellulose phthalate, polyvinyl acetate phthalate,carboxymethylethylcellulose, and EUDRAGIT®-type polymer(poly(methacrylic acid, methylmethacrylate), hydroxypropylmethylcellulose acetate succinate, cellulose acetate trimellitate,shellac or other suitable enteric coating polymers. The EUDRAGIT®-typepolymer include, for example, EUDRAGIT® FS 30D, L 30 D-55, L 100-55, L100, L 12,5, L 12,5 P, RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO,RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5, and S 12,5 P.In some embodiments, one or more of EUDRAGIT® FS 30D, L 30 D-55, L100-55, L 100, L 12,5, L 12,5 P RL 30 D, RL PO, RL 100, RL 12,5, RS 30D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5 andS 12,5 P is used. The enteric agent may be a combination of theforegoing solutions or dispersions.

In one embodiment, the modified-release formulation may include one ormore delayed-release coating(s) which remain essentially intact, or maybe essentially insoluble, in gastric fluid. The stability of thedelayed-release coating can be pH dependent. Delayed-release coatingsthat are pH dependent will be substantially stable in acidicenvironments (pH of about 5 or less), and substantially unstable in nearneutral to alkaline environments (pH greater than about 5). For example,the delayed-release coating may essentially disintegrate or dissolve innear neutral to alkaline environments such as are found in the smallintestine (e.g. one or more of the duodenum, jejunum, and ileum) and/orlarge intestine (e.g. one or more of the cecum, ascending colon,transverse colon, descending colon, and sigmoid colon).

By way of non-limiting example, there are various EUDRAGIT formulationsthat dissolve at rising pH, with formulations that dissolve at pH >5.5(EUDRAGIT L30 D-550), pH >6.0 (EUDRAGIT L12, 5), and pH >7.0 (EUDRAGITFS 30D). Since the ileum has the highest pH in the small intestine,ranging from 7.3 to 7.8, the use of EUDRAGIT FS 30D to coat the pelletcontaining the antibiotic-degrading enzyme, may delay the dissolution ofthe pellet until it reaches the ileum thereby localizing the release ofthe antibiotic-degrading enzyme to the ileum. However, the jejunum has apH ranging from 6.6 to 7.4, therefore, the release may initiate in somepatients in the jejunum, if the pH is at 7.0 or above. In suchembodiments, the antibiotic-degrading enzyme may be delivered with anantibiotic/inhibitor combination as described. The different types ofEUDRAGIT can be combined with each other, or multiple different types ofEUDRAGIT coatings can be combined to fine tune the dissolution profileto achieve targeted delivery to achieve optimal function. For example,EUDRAGIT L100, EUDRAGIT S100, and triethyl citrate may be mixed togetherat a ratio of, for example, about 72.7/18.2/9.1, to form a coating thatsubstantially releases at a pH of greater than about 6.2. In anotherexample, EUDRAGIT L100, EUDRAGIT S100, and triethyl citrate may be mixedtogether at a ratio of, for example, about 30/60.9/9, to form a coatingthat substantially releases at a pH of greater than about 6.7. In afurther example, DUOCOAT (KUECEPT) that uses two coatings of entericpolymers (like EUDRAGIT), an outer layer, and an inner layer ofpartially neutralized enteric polymer and a buffer agent. The DUOCOATtechnology allows more rapid release of the therapeutic agent initiatedat the targeted pH compared to a single coating of the enteric polymer(Liu et al., 2010, European J. Pharmaceutics and Biopharmaceuticals47:311, the entire contents of all of which are incorporated herein byreference). Release was demonstrated to be targeted to the ileum and/orileoceacal junction in 10 healthy volunteers (Varum et al., 2013,European J. Pharmaceutics and Biopharmaceuticals 84:573, the entirecontents of all of which are incorporated herein by reference).

In another embodiment, the delayed-release coating may degrade as afunction of time when in aqueous solution without regard to the pHand/or presence of enzymes in the solution. Such a coating may comprisea water insoluble polymer. Its solubility in aqueous solution istherefore independent of the pH. The term “pH independent” as usedherein means that the water permeability of the polymer and its abilityto release pharmaceutical ingredients is not a function of pH and/or isonly very slightly dependent on pH. Such coatings may be used toprepare, for example, sustained release formulations. Suitable waterinsoluble polymers include pharmaceutically acceptable non-toxicpolymers that are substantially insoluble in aqueous media, e.g., water,independent of the pH of the solution. Suitable polymers include, butare not limited to, cellulose ethers, cellulose esters, or celluloseether-esters, i.e., a cellulose derivative in which some of the hydroxygroups on the cellulose skeleton are substituted with alkyl groups andsome are modified with alkanoyl groups. Examples include ethylcellulose, acetyl cellulose, nitrocellulose, and the like. Otherexamples of insoluble polymers include, but are not limited to, lacquer,and acrylic and/or methacrylic ester polymers, polymers or copolymers ofacrylate or methacrylate having a low quaternary ammonium content, ormixture thereof and the like. Other examples of insoluble polymersinclude EUDRAGIT RS®, EUDRAGIT RL®, and EUDRAGIT NE®. Insoluble polymersuseful in the present invention include polyvinyl esters, polyvinylacetals, polyacrylic acid esters, butadiene styrene copolymers, and thelike. In one embodiment, colonic delivery is achieved by use of aslowly-eroding wax plug (e.g., various PEGS, including for example,PEG6000) or pectin. In an embodiment, the present invention contemplatesthe use of a delayed-release coating that degrade as a function of timewhich comprises a swell layer comprising croscarmellos sodium andhydroxyproplycellulose. In such embodiment, the formulation may furtherinclude an osmotic rupture coating that comprises ethylcellulose such asethylcellulose dispersions.

Alternatively, the stability of the modified-release formulation can beenzyme-dependent. Delayed-release coatings that are enzyme dependentwill be substantially stable in fluid that does not contain a particularenzyme and substantially unstable in fluid containing the enzyme. Thedelayed-release coating will essentially disintegrate or dissolve influid containing the appropriate enzyme. Enzyme-dependent control can bebrought about, for example, by using materials which release the activeingredient only on exposure to enzymes in the intestine, such asgalactomannans. Also, the stability of the modified-release formulationcan be dependent on enzyme stability in the presence of a microbialenzyme present in the gut flora. For example, in various embodiments,the delayed-release coating may be degraded by a microbial enzymepresent in the gut flora. In an embodiment, the delayed-release coatingmay be degraded by a bacteria present in the small intestine. In anotherembodiment, the delayed-release coating may be degraded by a bacteriapresent in the large intestine.

In various embodiments, the modified-release formulations of the presentinvention are designed for immediate release (e.g. upon ingestion). Invarious embodiments, the modified-release formulations may havesustained-release profiles, i.e. slow release of the activeingredient(s) in the body (e.g., GI tract) over an extended period oftime. In various embodiments, the modified-release formulations may havea delayed-release profile, i.e. not immediately release the activeingredient(s) upon ingestion; rather, postponement of the release of theactive ingredient(s) until the composition is lower in thegastrointestinal tract; for example, for release in the small intestine(e.g., one or more of duodenum, jejunum, ileum) or the large intestine(e.g., one or more of cecum, ascending, transverse, descending orsigmoid portions of the colon, and rectum). For example, a compositioncan be enteric coated to delay release of the active ingredient(s) untilit reaches the small intestine or large intestine. In some embodiments,there is not a substantial amount of the active ingredient(s) of thepresent formulations in the stool.

In various embodiments, the modified release formulation is designed forrelease in the colon. Various colon-specific delivery approaches may beutilized. For example, the modified release formulation may beformulated using a colon-specific drug delivery system (CODES) asdescribed for example, in Li et al., AAPS PharmSciTech (2002), 3(4):1-9, the entire contents of which are incorporated herein by reference.Drug release in such a system is triggered by colonic microflora coupledwith pH-sensitive polymer coatings. For example, the formulation may bedesigned as a core tablet with three layers of polymer. The firstcoating is an acid-soluble polymer (e.g., EUDRAGIT E), the outer coatingis enteric, along with an hydroxypropyl methylcellulose barrier layerinterposed in between. In another embodiment, colon delivery may beachieved by formulating the beta-lactamase (and/or additionaltherapeutic agent) with specific polymers that degrade in the colon suchas, for example, pectin. The pectin may be further gelled or crosslinkedwith a cation such as a zinc cation. In an embodiment, the formulationis in the form of ionically crosslinked pectin beads which are furthercoated with a polymer (e.g., EUDRAGIT polymer). Additional colonspecific formulations include, but are not limited to,pressure-controlled drug delivery systems (prepared with, for example,ethylcellulose) and osmotic controlled drug delivery systems (i.e.,ORDS-CT).

Formulations for colon specific delivery of beta-lactamases (and/oradditional therapeutic agents), as described herein, may be evaluatedusing, for example, in vitro dissolution tests. For example, paralleldissolution studies in different buffers may be undertaken tocharacterize the behavior of the formulations at different pH levels.Alternatively, in vitro enzymatic tests may be carried out. For example,the formulations may be incubated in fermenters containing suitablemedium for bacteria, and the amount of drug released at different timeintervals is determined. Drug release studies can also be done in buffermedium containing enzymes or rat or guinea pig or rabbit cecal contentsand the amount of drug released in a particular time is determined. In afurther embodiment, in vivo evaluations may be carried out using animalmodels such as dogs, guinea pigs, rats, and pigs. Further, clinicalevaluation of colon specific drug delivery formulations may be evaluatedby calculating drug delivery index (DDI) which considers the relativeratio of RCE (relative colonic tissue exposure to the drug) to RSC(relative amount of drug in blood i.e. that is relative systemicexposure to the drug). Higher drug DDI indicates better colon drugdelivery. Absorption of drugs from the colon may be monitored bycolonoscopy and intubation.

In various embodiments, the present formulation provide for substantialuniform dissolution of the beta-lactamase (and/or additional therapeuticagent) in the area of release in the GI tract. In an embodiment, thepresent formulation minimizes patchy or heterogeneous release of thebeta-lactamase. For example, when releasing in the distal smallintestine or, especially the colon, the distribution of beta-lactamase(and/or additional therapeutic agent) may be heterogeneous and thereforerequire formulation to minimize local effects.

In some embodiments, a dual pulse formulation is provided. In variousembodiments, the present invention provides for modified-releaseformulations that release multiple doses of the beta-lactamase, atdifferent locations along the intestines, at different times, and/or atdifferent pH. In an illustrative embodiment, the modified-releaseformulation comprises a first dose of the beta-lactamase and a seconddose of the beta-lactamase, wherein the first dose and the second doseare released at different locations along the intestines, at differenttimes, and/or at different pH. For example, the first dose is releasedat the duodenum, and the second dose is released at the ileum. Inanother example, the first dose is released at the jejunum, and thesecond dose is released at the ileum. In other embodiments, the firstdose is released at a location along the small intestine (e.g., theduodenum), while the second dose is released along the large intestine(e.g., the ascending colon). In various embodiments, themodified-release formulation may release at least one dose, at least twodoses, at least three doses, at least four doses, at least five doses,at least six doses, at least seven doses, or at least eight doses of thebeta-lactamase at different locations along the intestines, at differenttimes, and/or at different pH. Further the dual pulse description hereinapplies to modified-release formulations that release a beta-lactamaseand an additional therapeutic agent.

In some embodiments, a dual pulse formulation is provided in which adose of the beta-lactamase and a dose of an oral antibiotic (and/or abeta-lactamase inhibitor) are released at different locations along theintestines, at different times, and/or at different pH. For example, thedose of an oral antibiotic (and/or a beta-lactamase inhibitor) isreleased proximal to the dose of the beta-lactamase. For example, thedose of an oral antibiotic (and/or a beta-lactamase inhibitor) isreleased in the ileum and upstream and the dose of the beta-lactamase isreleased in the distal small intestine and/or the colon.

In various embodiments, the invention provides a formulation comprising:a core particle having a base coat comprising one or morebeta-lactamases, and a delayed-release coating disposed over the coatedcore particle. The delayed-release coating may be substantially stablein acidic environments and/or gastric fluid, and/or substantiallyunstable in near neutral to alkaline environments or intestinal fluidthereby exposing the coated core particle to intestinal fluid. The basecoat comprising one or more beta-lactamases may further comprise one ormore additional therapeutic agents. Optionally a plurality of base coatsmay be applied to the core particle each of which may contain abeta-lactamase and/or an additional therapeutic agent. In an embodiment,the core particle includes sucrose.

In an embodiment, a beta-lactamases can be sprayed onto an inert core(e.g., a sucrose core or a cellulose core such as a microcrystallinesucrose or cellulose core) and spray-dried with an enteric layer to formbeta-lactamase containing pellets or beads. In various embodiments, theenteric layer may comprise one or more enteric agents as describedherein. For example, the enteric layer may comprise an EUDRAGIT®-typepolymer such as EUDRAGIT L30 D-55, as described for example, inPCT/US2015/054606, the entire disclosure of which is hereby incorporatedby reference. In such an embodiment, the formulation comprising thebeta-lactamase containing pellets or beads may release thebeta-lactamase at a pH of about 5.5.

In an embodiment, the enteric layer may comprise a mixture ofEUDRAGIT®-type polymers. In various embodiments, the enteric layer maycomprise a mixture of EUDRAGIT L100, EUDRAGIT S100, and triethylcitrate. In some embodiments, the enteric layer comprises about 65% toabout 85% of EUDRAGITL100, about 10% to about 30% of EUDRAGIT S100, andabout 1% to about 20% of triethyl citrate. In an embodiment, the entericlayer comprises about 73% of EUDRAGITL100, about 18% of EUDRAGIT S100,and about 9% of triethyl citrate. In an embodiment, the enteric layercomprises about 72.7% of EUDRAGITL100, about 18.2% of EUDRAGIT S100, andabout 9.1% of triethyl citrate. In such embodiments, the formulationcomprising the beta-lactamase containing pellets or beads may releasethe beta-lactamase at a pH of greater than about 6.2. In variousembodiments, the beta-lactamase containing pellets or beads isspray-dried with an enteric layer having a coating weight of about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. Inan embodiment, the beta-lactamase containing pellets or beads isspray-dried with an enteric layer having a coating weight of about 35%.In various embodiments, the beta-lactamase containing pellets or beadsis spray-dried with an enteric layer having a coating thickness of about50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM,about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about105 μM, about 110 μM, about 115 μM, or about 120 μM. In an embodiment,the beta-lactamase containing pellets or beads is spray-dried with anenteric layer having a coating thickness of about 100 μM. In anembodiment, the beta-lactamase containing pellets or beads isspray-dried with an enteric layer having a coating thickness of about99.7 μM.

In some embodiments, the enteric layer comprises about 20% to about 40%of EUDRAGITL100, about 50% to about 70% of EUDRAGIT S100, and about 1%to about 20% of triethyl citrate. In an embodiment, the enteric layercomprises about 30% of EUDRAGITL100, about 61% of EUDRAGIT S100, andabout 9% of triethyl citrate.

In an embodiment, the enteric layer comprises about 30% of EUDRAGITL100,about 60.9% of EUDRAGIT S100, and about 9.1% of triethyl citrate. Insuch embodiments, the formulation comprising the beta-lactamasecontaining pellets or beads may release the beta-lactamase at a pH ofgreater than about 6.7. In various embodiments, the beta-lactamasecontaining pellets or beads is spray-dried with an enteric layer havinga coating weight of about 20%, about 25%, about 30%, about 35%, about40%, about 45%, or about 50%. In an embodiment, the beta-lactamasecontaining pellets or beads is spray-dried with an enteric layer havinga coating weight of about 35%. In various embodiments, thebeta-lactamase containing pellets or beads is spray-dried with anenteric layer having a coating thickness of about 50 μM, about 55 μM,about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about85 μM, about 90 μM, about 95 μM, about 100 μM, about 105 μM, about 110μM, about 115 μM, or about 120 μM. In an embodiment, the beta-lactamasecontaining pellets or beads is spray-dried with an enteric layer havinga coating thickness of about 110 μM. In an embodiment, thebeta-lactamase containing pellets or beads is spray-dried with anenteric layer having a coating thickness of about 113 μM.

In various embodiments, the invention provides a formulation comprisinga delayed-release coating that releases the beta-lactamase in apH-independent manner and/or a time-dependent manner. In variousembodiments, the formulation comprises: a core particle having a basecoat comprising one or more beta-lactamases, a swell layer, and anosmotic rupture coating disposed over the coated core particle with theswell layer. In an embodiment, a beta-lactamases can be sprayed onto aninert core (e.g., a sucrose core or a cellulose core such as amicrocrystalline sucrose or cellulose core) with the swell layer and theosmotic rupture coating added subsequently.

In such embodiments, the delayed-release coating including the swelllayer and the osmotic rupture coating allows for release of thebeta-lactamase within a specified time frame. In various embodiments,the beta-lactamase is released after about 2 hours, about 2.5 hours,about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours,about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about9.5 hours, or about 10 hours after ingestion. In various embodiments,the swell layer comprises about 60% to about 80% of croscarmellos sodium(e.g., AcDiSol) and about 20% to about 40% of hydroxyproplycellulose(HPC). In an embodiment, the swell layer comprises about 71% pulverizedcroscarmellos sodium and about 29% hydroxyproplycellulose. In anembodiment, the swell layer comprises about 71.4% pulverizedcroscarmellos sodium and about 28.6% hydroxyproplycellulose. In variousembodiments, the osmotic rupture coating comprises about 65% to about85% of ethylcellulose dispersion (e.g., Aquacoat ECD) and about 15% toabout 35% triethyl citrate. In an embodiment, the osmotic rupturecoating comprises about 75% ethylcellulose dispersion and about 25%triethyl citrate. In various embodiments, the beta-lactamase containingpellets or beads with the swell layer and osmotic rupture coating has acoating weight of about 5%, about 10%, about 15%, about 20%, about 25%,or about 30%. In an embodiment, the beta-lactamase containing pellets orbeads with the swell layer and osmotic rupture coating has a coatingweight of about 13.5%.

In various embodiments, the beta-lactamase containing pellets or beadswith the swell layer and osmotic rupture coating has a coating thicknessof about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM,about 45 μM, about 50 μM, about 55 μM, or about 60 μM. In an embodiment,the beta-lactamase containing pellets or beads with the swell layer andosmotic rupture coating has a coating thickness of about 50 μM. In anembodiment, the beta-lactamase containing pellets or beads with theswell layer and osmotic rupture coating has a coating thickness of about48 μM.

Optionally, the core particle may comprise one or more beta-lactamasesand/or one or more additional therapeutic agents. In one embodiment, oneor more doses of the beta-lactamase may be encapsulated in a coreparticle, for example, in the form of a microsphere or a mini-sphere.For example, the beta-lactamase may be combined with a polymer (e.g.,latex), and then formed into a particulate, micro-encapsulated enzymepreparation, without using a sucrose core. The microspheres ormini-spheres thus formed may be optionally covered with adelayed-release coating.

A variety of approaches for generating particulates (such asmicrospheres, mini-spheres, aggregates, other) may be utilized for theinclusion of enzymatic proteins. They typically involve at least twophases, one containing the protein, and one containing a polymer thatforms the backbone of the particulate. Most common are coacervation,where the polymer is made to separate from its solvent phase by additionof a third component, or multiple phase emulsions, such as water in oilin water (w/o/w) emulsion where the inner water phase contains theprotein, the intermediate organic phase contains the polymer, and theexternal water phase stabilizers that support the w/o/w double emulsionuntil the solvents can be removed to form, for example, microspheres ormini-spheres. Alternatively, the beta-lactamase and stabilizingexcipients (for example, trehalose, mannitol, Tween 80, polyvinylalcohol) are combined and sprayed from aqueous solution and collected.The particles are then suspended in a dry, water immiscible organicsolvent containing polymer and release modifying compounds, and thesuspension sonicated to disperse the particles. An additional approachuses aqueous phases but no organic solvent. Specifically, the enzymaticprotein, buffer components, a polymer latex, and stabilizing andrelease-modifying excipients are dissolved/dispersed in water. Theaqueous dispersion is spray-dried, leading to coalescence of the latex,and incorporation of the protein and excipients in particles of thecoalesced latex. When the release modifiers are insoluble at acidicconditions but soluble at higher pHs (such as carboxylic acid) thenrelease from the matrix is inhibited in the gastric environment. In anembodiment, the beta-lactamase may be initially solubilized as anemulsion, microemulsion, or suspension and then formulated into solidmini-spheres or microspheres. The formulation may then be coated with,for example, a delayed-release, sustained-release, or controlled-releasecoating to achieve delivery at a specific location such as, for example,the intestines.

In various embodiments, the formulation may comprise a plurality ofmodified-release particles or beads or pellets or microspheres. In anembodiment, the formulation is in the form of capsules comprisingmultiple beads. In another embodiment, the formulation is in the form ofcapsules comprising multiple pellets. In another embodiment, theformulation is in the form of capsules comprising multiple microspheresor mini-spheres.

In some embodiments, before applying the delayed-release coating to thecoated core particle, the particle can optionally be covered with one ormore separating layers comprising pharmaceutical excipients includingalkaline compounds such as for instance pH-buffering compounds. Theseparating layer essentially separates the coated core particle from thedelayed-release coating.

The separating layer can be applied to the coated core particle bycoating or layering procedures typically used with coating equipmentsuch as a coating pan, coating granulator or in a fluidized bedapparatus using water and/or organic solvents for the coating process.As an alternative the separating layer can be applied to the corematerial by using a powder coating technique. The materials forseparating layers are pharmaceutically acceptable compounds such as, forinstance, sugar, polyethylene glycol, polyvinylpyrrolidone, polyvinylalcohol, polyvinyl acetate, hydroxypropyl cellulose, methyl-cellulose,ethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulosesodium and others, used alone or in mixtures. Additives such asplasticizers, colorants, pigments, fillers, anti-tacking and anti-staticagents, such as for instance magnesium stearate, sodium stearylfumarate, titanium dioxide, talc and other additives can also beincluded in the separating layer.

In some embodiments, the coated particles with the delayed-releasecoating may be further covered with an overcoat layer. The overcoatlayer can be applied as described for the other coating compositions.The overcoat materials are pharmaceutically acceptable compounds such assugar, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol,polyvinyl acetate, hydroxypropyl cellulose, methylcellulose,ethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulosesodium and others, used alone or in mixtures. The overcoat materials canprevent potential agglomeration of particles coated with thedelayed-release coating, protect the delayed-release coating fromcracking during the compaction process or enhance the tableting process.

In various embodiments, the formulation may comprise a plurality ofmodified-release particles or pellets or microspheres. In oneembodiment, the formulation is in the form of capsules comprisingmultiple pellets. In one embodiment, the formulation is in the form ofcapsules comprising multiple microspheres.

In some embodiments, the modified-release formulation is a capsulefilled with a plurality of beta-lactamase-containing pellets (e.g., P3A(or the other beta-lactamase agents described, herein, and variantsthereof)-containing pellets) from which the beta-lactamase is released.In an embodiment, the capsule is a gelatin capsule, such as a hardgelatin capsule. In another embodiment, the capsule is a hydroxypropylmethylcellulose (HPMC) capsule. For example, the formulation may be inthe form of capsules comprising multiple pellets. For example, theformulation may be in the form of capsules such as, for example, gelatinor hydroxypropyl methylcellulose (HPMC) capsules comprising multipleenteric-coated pellets containing beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof). Inanother example, the formulation may be in the form of capsules such as,for example, gelatin or hydroxypropyl methylcellulose (HPMC) capsulescomprising multiple pellets containing beta-lactamase (e.g. P3A, or theother beta-lactamase agents described herein, and variants thereof)coated with an osmotic-rupture coating. In such embodiments, acombination of pellets may be utilized in which each pellet is designedto release at a specific time point or location. In various embodiments,the pellets (e.g., enteric-coated pellets) are designed to pass throughthe stomach unchanged and then release the beta-lactamase (e.g. P3A, orthe other beta-lactamase agents described herein, and variants thereof)into one or more regions of the intestines. In some embodiments, thebeta-lactamase-containing pellets may be enteric-coated to release thebeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof) at different intestinal pH values. In someembodiments, the beta-lactamase-containing pellets may be coated with anosmotic-rupture coating to release the beta-lactamase (e.g. P3A, or theother beta-lactamase agents described herein, and variants thereof)within specific time frames.

In various embodiments, the formulation of the present invention is inthe form of a capsule (e.g., a hard gelatin or HPMC capsule) comprisinga plurality of enteric-coated beta-lactamase-containing pellets. In suchembodiments, the pellets (or each individual pellet) comprise abeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof) and a sucrose sphere, which thebeta-lactamase, for example, P3A or a variant, is sprayed onto, acoating comprising for example one or more enteric polymers, and/oradditional excipients and/or buffer salts. For example, the pellets maycomprise a binder excipient (e.g., hydroxypropylcellulose (HPC) orhydroxypropyl methylcellulose (HPMC)), one or more enteric polymers(e.g., EUDRAGIT L 30 D-55, EUDRAGIT L100, EUDRAGIT S100), a plasticizer(e.g., triethyl citrate), and buffer salts.

In various embodiments, the formulation of the present invention is inthe form of a capsule (e.g., a hard gelatin or HPMC capsule) comprisinga plurality of enteric-coated beta-lactamase-containing pellets. In suchembodiments, the pellets (or each individual pellet) comprise about10-20% by weight of beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof). Forexample, the beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof) may be present at about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 19%, or about 20% by weight. In someembodiments, the pellets (or each individual pellet) comprise about10-25% by weight sucrose sphere, which the beta-lactamase, for example,P3A or a variant, is sprayed onto. For example, the sucrose sphere maybe present at about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about21%, about 22%, about 23%, about 24%, or about 25% by weight. In variousembodiments, the pellets (or each individual pellet) comprise about20-35% by weight a binder excipient (e.g., hydroxypropylcellulose (HPC)or hydroxypropyl methylcellulose (HPMC)). For example, the binderexcipient may be present at about 20%, about 21%, about 22%, about 23%,about 24%, or about 25%, about 26%, about 27%, about 28%, about 29%,about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% byweight. In some embodiments, the pellets (or each individual pellet)comprise about 10-30% by weight of a first enteric polymer (e.g.,EUDRAGIT L100). For example, the first enteric polymer (e.g., EUDRAGITL100) may be present at about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, or about 30% by weight. In someembodiments, the pellets (or each individual pellet) comprise about1-30% by weight of a second enteric polymer (e.g., EUDRAGIT S100). Forexample, the second enteric polymer (e.g., EUDRAGIT S100) may be presentat about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about27%, about 28%, about 29%, or about 30% by weight. In some embodiments,the pellets (or each individual pellet) comprise about 1-10% by weightof plasticizer (e.g., triethyl citrate). For example, the plasticizermay be present at about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, or about 10% by weight. In someembodiments, the pellets (or each individual pellet) further compriseabout 1-2% by weight buffer salts. For example, the buffer salts may bepresent at about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%,about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% by weight.The weight as described herein refers to the total weight of allcomponents excluding the weight of the capsule itself.

In some embodiments, the pellets (or each individual pellet) compriseabout 14% by weight of the beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof); about 21%by weight sucrose sphere; about 31% by weight a binder excipient (e.g.,hydroxypropylcellulose (HPC) or hydroxypropyl methylcellulose (HPMC));about 24% by weight a first enteric polymer (e.g., EUDRAGIT L100); about6% by weight a second enteric polymer (e.g., EUDRAGIT S100); about 3% byweight of plasticizer (e.g., triethyl citrate); and about 1% by weightbuffer salts. The weight as described herein refers to the total weightof all components excluding the weight of the capsule itself. In suchembodiments, the pellets may release the beta-lactamase at a pH ofgreater than about 6, about 6.1, about 6.2, about 6.3, about 6.4, orabout 6.5. In an embodiment, the pellets may release the beta-lactamaseat a pH of greater than about 6.2.

For example, the pellets (or each individual pellet) comprise about13.9% by weight of the beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof); about20.5% by weight sucrose sphere; about 30.7% by weight a binder excipient(e.g., hydroxypropylcellulose (HPC)); about 24.3% by weight a firstenteric polymer (e.g., EUDRAGIT L100); about 6.1% by weight a secondenteric polymer (e.g., EUDRAGIT S100); about 3% by weight of plasticizer(e.g., triethyl citrate); and about 1.4% by weight buffer salts. Theweight as described herein refers to the total weight of all componentsexcluding the weight of the capsule itself. In an embodiment, thepellets may release the beta-lactamase at a pH of greater than about6.2.

In some embodiments, the pellets (or each individual pellet) compriseabout 13% by weight of the beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof); about 18%by weight sucrose sphere; about 28% by weight a binder excipient (e.g.,hydroxypropylcellulose (HPC) or hydroxypropyl methylcellulose (HPMC));about 12% by weight a first enteric polymer (e.g., EUDRAGIT L100); about25% by weight a second enteric polymer (e.g., EUDRAGIT S100); about 4%by weight of plasticizer (e.g., triethyl citrate); and about 1% byweight buffer salts. The weight as described herein refers to the totalweight of all components excluding the weight of the capsule itself. Insuch embodiments, the pellets may release the beta-lactamase at a pH ofgreater than about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, orabout 7.0. In an embodiment, the pellets may release the beta-lactamaseat a pH of greater than about 6.7.

For example, the pellets (or each individual pellet) comprise about12.5% by weight of the beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof); about18.4% by weight sucrose sphere; about 27.5% by weight a binder excipient(e.g., hydroxypropylcellulose (HPC)); about 12.2% by weight a firstenteric polymer (e.g., EUDRAGIT L100); about 24.5% by weight a secondenteric polymer (e.g., EUDRAGIT S100); about 3.7% by weight ofplasticizer (e.g., triethyl citrate); and about 1.3% by weight buffersalts. The weight as described herein refers to the total weight of allcomponents excluding the weight of the capsule itself. In theembodiment, the pellets may release the beta-lactamase at a pH ofgreater than about 6.7.

In various embodiments, the formulation of the present invention is inthe form of a capsule (e.g., a hard gelatin or HPMC capsule) comprisingabout 50 mg of the beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof). The capsule includes aplurality of enteric-coated beta-lactamase-containing pellets. In suchembodiments, the formulation comprises about 5-15% by weight of thebeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof). For example, the beta-lactamase (e.g.P3A, or the other beta-lactamase agents described herein, and variantsthereof) may be present at about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%by weight. In some embodiments, the formulation comprises about 10-20%by weight sucrose sphere. For example, the sucrose sphere may be presentabout 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, or about 20% by weight. In variousembodiments, the formulation comprises about 20-30% by weight a binderexcipient (e.g., hydroxypropylcellulose (HPC) or hydroxypropylmethylcellulose (HPMC)). For example, the binder excipient may bepresent at about 20%, about 21%, about 22%, about 23%, about 24%, about25%, about 26%, about 27%, about 28%, about 29%, or about 30% by weight.In some embodiments, the formulation comprises about 5-25% by weight afirst enteric polymer (e.g., EUDRAGIT L100). For example, the firstenteric polymer may be present at about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about21%, about 22%, about 23%, about 24%, or about 25% by weight. In someembodiments, the formulation comprises about 1-25% by weight a secondenteric polymer (e.g., EUDRAGIT S100). For example, the second entericpolymer may be present at about 1%, about 2%, about 3%, about 4%, about5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%,about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, orabout 25% by weight. In some embodiments, the formulation comprisesabout 1-10% by weight of plasticizer (e.g., triethyl citrate). Forexample, the plasticizer may be present at about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%by weight. In some embodiments, the formulation comprises about 0.5-1.5%by weight buffer salts. For example, the buffer salts may be present atabout 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.1%,about 1.2%, about 1,3%, about 1.4%, or about 1.5% by weight. In someembodiments, the formulation comprises about 15-25% by weight gelatin orHPMC capsule. For example, the gelatin or HPMC capsule may be about 15%,about 16%, about 17%, about 18%, about 19%, about 20% about 21%, about22%, about 23%, about 24%, or about 25% by weight.

In some embodiments, the formulation of the present invention comprisesabout 50 mg of the beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof). In such embodiments, theformulation comprises about 11% by weight of the beta-lactamase (e.g.P3A, or the other beta-lactamase agents described herein, and variantsthereof); about 17% by weight sucrose sphere; about 25% by weight abinder excipient (e.g., hydroxypropylcellulose (HPC)); about 20% byweight a first enteric polymer (e.g., EUDRAGIT L100); about 5% by weighta second enteric polymer (e.g., EUDRAGIT S100); about 2% by weight ofplasticizer (e.g., triethyl citrate); about 1% by weight buffer salts;and about 21% by weight gelatin or HPMC capsule. In such embodiments,the pellets within the capsule may release the beta-lactamase at a pH ofgreater than about 6, about 6.1, about 6.2, about 6.3, about 6.4, orabout 6.5. In an embodiment, the pellets may release the beta-lactamaseat a pH of greater than about 6.2.

For example, the formulation comprises about 11.2% by weight of thebeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof); about 16.6% by weight sucrose sphere;about 24.8% by weight a binder excipient (e.g., hydroxypropylcellulose(HPC)); about 19.7% by weight a first enteric polymer (e.g., EUDRAGITL100); about 4.9% by weight a second enteric polymer (e.g., EUDRAGITS100); about 2.4% by weight of plasticizer (e.g., triethyl citrate);about 1.1% by weight buffer salts; and about 20.9% by weight gelatin orHPMC capsule. In an embodiment, the pellets within the capsule mayrelease the beta-lactamase at a pH of greater than about 6.2.

In some embodiments, the formulation of the present invention comprisesabout 50 mg of the beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof). In such embodiments, theformulation comprises about 10% by weight of the beta-lactamase (e.g.P3A, or the other beta-lactamase agents described herein, and variantsthereof); about 15% by weight sucrose sphere; about 22% by weight abinder excipient (e.g., hydroxypropylcellulose (HPC)); about 10% byweight a first enteric polymer (e.g., EUDRAGIT L100); about 20% byweight a second enteric polymer (e.g., EUDRAGIT S100); about 3% byweight of plasticizer (e.g., triethyl citrate); about 1% by weightbuffer salts; and about 19% by weight gelatin or HPMC capsule. In suchembodiments, the pellets within the capsule may release thebeta-lactamase at a pH of greater than about 6.5, about 6.6, about 6.7,about 6.8, about 6.9, or about 7.0. In an embodiment, the pellets mayrelease the beta-lactamase at a pH of greater than about 6.7.

For example, the formulation comprises about 10.1% by weight of thebeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof); about 14.9% by weight sucrose sphere;about 22.4% by weight a binder excipient (e.g., hydroxypropylcellulose(HPC)); about 9.9% by weight a first enteric polymer (e.g., EUDRAGITL100); about 19.9% by weight a second enteric polymer (e.g., EUDRAGITS100); about 3% by weight of plasticizer (e.g., triethyl citrate); about1% by weight buffer salts; and about 18.8% by weight gelatin or HPMCcapsule. In an embodiment, the pellets within the capsule may releasethe beta-lactamase at a pH of greater than about 6.7.

In various embodiments, the formulation of the present invention is inthe form of a capsule (e.g., a hard gelatin or HPMC capsule) comprisinga plurality of beta-lactamase-containing pellets which are coated with aswelling layer and/or an osmotic rupture coating. In such embodiments,the pellets (or each individual pellet) comprise a beta-lactamase (e.g.P3A, or the other beta-lactamase agents described herein, and variantsthereof) and a sucrose sphere, which the beta-lactamase, for example,P3A or a variant, is sprayed onto, one or more coatings comprising forexample one or more swelling layers and/or osmotic rupture coatings,and/or additional excipients and/or buffer salts. For example, thepellets may comprise a binder excipient (e.g., hydroxypropylcellulose(HPC) or hydroxypropyl methylcellulose (HPMC)), one or more swellinglayers comprising, for example, croscarmellos sodium (e.g., pulverizedcroscarmellos sodium such as AcDiSol), one or more osmotic rupturecoatings comprising, for example, ethylcellulose (e.g., ethylcellulosedispersions such as Aquacoat ECD), one or more excipient s (e.g., talc),and buffer salts.

In various embodiments, the formulation of the present invention is inthe form of a capsule (e.g., a hard gelatin or HPMC capsule) comprisinga plurality of beta-lactamase-containing pellets coated with a swellinglayer and/or an osmotic rupture coating. In such embodiments, thepellets (or each individual pellet) comprise about 5-15% by weight ofbeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof). For example, the beta-lactamase (e.g.P3A, or the other beta-lactamase agents described herein, and variantsthereof) may be present at about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%; about 13%, about 14%, or about 15%by weight. In some embodiments, the pellets (or each individual pellet)comprise about 10-20% by weight sucrose sphere, which thebeta-lactamase, for example, P3A or a variant, is sprayed onto. Forexample, the sucrose sphere may be present at about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, or about 20% by weight. In various embodiments, thepellets (or each individual pellet) comprise about 25-35% by weight abinder excipient (e.g., hydroxypropylcellulose (HPC) or hydroxypropylmethylcellulose (HPMC)). For example, the binder excipient may bepresent at about 25%, about 26%, about 27%, about 28%, about 29%, about30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight.In some embodiments, the pellets (or each individual pellet) comprise aswelling layer comprising pulverized croscarmellos sodium (e.g.,AcDiSol), which is about 20-30% by weight. For example, the pulverizedcroscarmellos sodium (e.g., AcDiSol) may be present at about 20%, about21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%,about 28%, about 29%, or about 30% by weight. In some embodiments, thepellets (or each individual pellet) comprise an osmotic rupture coatingcomprising ethylcellulose dispersion (e.g., Aquacoat ECD), which isabout 1-10% by weight. For example, the ethylcellulose dispersion (e.g.,Aquacoat ECD) may be present at about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% byweight. In some embodiments, the pellets (or each individual pellet)comprise about 1-10% by weight of an excipient (e.g., talc). Forexample, the excipient may be present at about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%by weight. In some embodiments, the pellets (or each individual pellet)further comprise about 0.5-1.5% by weight buffer salts. For example, thebuffer salts may be present at about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, orabout 1.5% by weight. The weight as described herein refers to the totalweight of all components excluding the weight of the capsule itself.

In some embodiments, the pellets (or each individual pellet) compriseabout 11% by weight of the beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof); about 17%by weight sucrose sphere; about 31% by weight a binder excipient (e.g.,hydroxypropylcellulose (HPC) or hydroxypropyl methylcellulose (HPMC));about 25% by weight pulverized croscarmellos sodium (e.g., AcDiSol);about 7% by weight ethylcellulose dispersion (e.g., Aquacoat ECD); about9% by weight of an excipient (e.g., talc); and about 1% by weight buffersalts. The weight as described herein refers to the total weight of allcomponents excluding the weight of the capsule itself.

For example, the pellets (or each individual pellet) comprise about11.2% by weight of the beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof); about16.5% by weight sucrose sphere; about 31.3% by weight a binder excipient(e.g., hydroxypropylcellulose (HPC) or hydroxypropyl methylcellulose(HPMC)); about 24.8% by weight pulverized croscarmellos sodium (e.g.,AcDiSol); about 6.6% by weight ethylcellulose dispersion (e.g., AquacoatECD); about 8.6% by weight of an excipient (e.g., talc); and about 1.1%by weight buffer salts. The weight as described herein refers to thetotal weight of all components excluding the weight of the capsuleitself. The weight as described herein refers to the total weight of allcomponents excluding the weight of the capsule itself.

In various embodiments, the formulation of the present invention is inthe form of a capsule (e.g., a hard gelatin or HPMC capsule) comprisingabout 50 mg of the beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof). The capsule includes aplurality of beta-lactamase-containing pellets coated with a swellinglayer and/or an osmotic rupture coating. In such embodiments, theformulation comprises about 5-15% by weight of the beta-lactamase (e.g.P3A, or the other beta-lactamase agents described herein, and variantsthereof). For example, the beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof) may bepresent at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, or about 15% by weight. Insome embodiments, the formulation comprises about 10-20% by weightsucrose sphere. For example, the sucrose sphere may be present about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 19%, or about 20% by weight. In someembodiments, the formulation comprises about 20-30% by weight a binderexcipient (e.g., hydroxypropylcellulose (HPC) or hydroxypropylmethylcellulose (HPMC)). For example, the binder excipient may bepresent at about 20%, about 21%, about 22%, about 23%, about 24%, about25%, about 26%, about 27%, about 28%, about 29%, or about 30% by weight.In some embodiments, the formulation comprises about 15-25% by weightcroscarmellos sodium (e.g., AcDiSol). For example, the croscarmellossodium (e.g., AcDiSol) may be present at about 15%, about 16%, about17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%,about 24%, or about 25% by weight. In some embodiments, the formulationcomprises about 1-10% by weight ethylcellulose dispersion (e.g.,Aquacoat ECD). For example, the ethylcellulose dispersion (e.g.,Aquacoat ECD) may be present at about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% byweight. In some embodiments, the formulation comprises about 1-10% byweight of an excipient (e.g., talc). For example, the excipient may bepresent at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, or about 10% by weight. In someembodiments, the formulation comprises about 0.5-1.5% by weight buffersalts. For example, the buffer salts may be present at about 0.5%, about0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.1%, about 1.2%, about1.3%, about 1.4%, or about 1.5% by weight. In some embodiments, theformulation comprises about 10-20% by weight gelatin or HPMC capsule.For example, the gelatin or HPMC capsule may be about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, or about 20% by weight.

In some embodiments, the formulation of the present invention comprisesabout 50 mg of the beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof). In such embodiments, theformulation-comprises about 9% by weight of the beta-lactamase (e.g.P3A, or the other beta-lactamase agents described herein, and variantsthereof); about 14% by weight sucrose sphere; about 26% by weight abinder excipient (e.g., hydroxypropylcellulose (HPC) or hydroxypropylmethylcellulose (HPMC)); about 20% by weight croscarmellos sodium (e.g.,AcDiSol); about 5% by weight ethylcellulose dispersion (e.g., AquacoatECD); about 7% by weight of an excipient (e.g., talc); about 1% byweight buffer salts; and about 17% by weight gelatin or HPMC capsule.

For example, the formulation may comprise about 9.3% by weight of thebeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof); about 13.7% by weight sucrose sphere;about 26% by weight a binder excipient (e.g., hydroxypropylcellulose(HPC) or hydroxypropyl methylcellulose (HPMC)); about 20.4% by weightcroscarmellos sodium (e.g., AcDiSol); about 5.4% by weightethylcellulose dispersion (e.g., Aquacoat ECD); about 7.1% by weight ofan excipient (e.g., talc); about 0.9% by weight buffer salts; and about17.3% by weight gelatin or HPMC capsule.

In some embodiments, the present formulations are those presented inTABLE 5.

In various embodiments, the formulations may combine a beta-lactamasewith a latex, or other polymer, and a particulate, micro-encapsulatedenzyme preparation will be formed. The microspheres then may be coveredwith a pH-dependent enteric coating. In some embodiments, no sucrosecore is required and this allows for higher drug loading per pellet andtherefore a smaller capsule size for therapy. There are a variety ofapproaches for generating particulates (such as microspheres,aggregates, other) that are amenable to the inclusion of proteins. Insome embodiments, the approaches involve at least two phases, onecontaining the protein, and one containing a polymer that forms thebackbone of the particulate. For example, one or more of the followingmay be used: coacervation, where the polymer is made to separate fromits solvent phase by addition of a third component, or multiple phaseemulsions, such as water in oil in water. (w/o/w) emulsion where theinner water phase contains the protein, the intermediate organic phasecontains the polymer, and the external water phase stabilizers thatsupport the w/o/w double emulsion until the solvents can be removed toform the microspheres.

In some embodiments, the protein and stabilizing excipients (e.g.,trehalose, mannitol, Tween 80, polyvinyl alcohol) are combined and thenthe mixture is sprayed from aqueous solution and particles that areformed are collected. The particles are then suspended in a dry, waterimmiscible organic solvent containing polymer and release modifyingcompounds, and the suspension sonicated to disperse the particles. It isanticipated that the enzyme will retain its activity following thisprocess. Another approach uses aqueous phases but no organic solvent.Here, the enzyme, buffer components, a polymer latex, and stabilizingand release-modifying excipients are dissolved/dispersed in water. Theaqueous dispersion is spray-dried, leading to coalescence of the latex,and incorporation of the protein and excipients in particles of thecoalesced latex. If the release modifiers are insoluble at acidicconditions but soluble at higher pHs (such as carboxylic acidic) thenrelease from the matrix should be inhibited in the gastric environment.

In various embodiments, the formulations of the present invention takethe form of those as described in U.S. Provisional Patent ApplicationNo. 62/061,507, the entire contents of all of which are incorporatedherein by reference.

In various embodiments, the formulations of the present invention takethe form of those as described in one or more of U.S. Pat. Nos.8,535,713 and 8,9117,77 and US Patent Publication Nos. 20120141585,20120141531, 2006/001896, 2007/0292523, 200810020018, 2008/0113031,2010/0203120, 2010/0255087, 2010/0297221, 2011/0052645, 2013/0243873,2013/0330411, 2014/0017313, and 2014/0234418, the contents of which arehereby incorporated by reference in their entirety.

In various embodiments, the formulations of the present invention takethe form of those as described in International Patent Publication No.WO 2008/135090, the contents of which are hereby incorporated byreference in their entirety.

In various embodiments, the formulations of the present invention takethe form of those described in one or more of U.S. Pat. Nos. 4,196,564;4,196,565; 4,247,006; 4,250,997; 4,268,265; 5,317,849; 6,572,892;7,712,634; 8,074,835; 8,398,912; 8,440,224; 8,557,294; 8,646,591;8,739,812; 8,810,259; 8,852,631; and 8,911,788 and US Patent PublicationNos. 2014/0302132; 2014/0227357; 20140088202; 20130287842; 2013/0295188;2013/0307962; and 20130184290 the contents of which are herebyincorporated by reference in their entirety.

In some embodiments, the present formulation includes about 0.1-1%beta-lactamase, about 0.1-1% pore former, about 5-15% matrix, about0.1-1% lubricant, about 0.1-1% buffer, about 0.5-5% protectant, andabout 80-90% water. In some embodiments, the present formulationincludes about 0.1-1% beta-lactamase (e.g. P3A), about 0.1-1% poreformer (e.g. HPMCAS-MF), about 5-15% matrix (e.g. Aquacoat (FMC)), about0.1-1% lubricant (e.g. Sodium-Stearyl Fumarate), about 0.1-1% buffer(e.g. Sodium Hydrogen Phosphate), about 0.5-5% protectant (e.g.Trehalose), and about 80-90% water.

In some embodiments, the present formulation includes about 0.5%beta-lactamase, about 0.5% pore former, about 10% matrix, about 0.5%lubricant, about 0.5% buffer, about 1% protectant, and about 90% water.In some embodiments, the present formulation includes about 0.5%beta-lactamase (e.g. P3A), about 0.5% pore former (e.g. HPMCAS-MF),about 10% matrix (e.g. Aquacoat (FMC)), about 0.5% lubricant (e.g.Sodium-Stearyl Fumarate), about 0.5% buffer (e.g. Sodium HydrogenPhosphate), about 1% protectant (e.g. Trehalose), and about 90% water.

In some embodiments, the present formulation includes about 0.5%beta-lactamase, about 0.3% pore former, about 10.1% matrix, about 0.2%lubricant, about 0.1% buffer, about 1% protectant, and about 88.8%water. In some embodiments, the present formulation includes about 0.5%beta-lactamase (e.g. P3A), about 0.3% pore former (e.g. HPMCAS-MF),about 10.1% matrix (e.g. Aquacoat (FMC)), about 0.2% lubricant (e.g.Sodium-Stearyl Fumarate), about 0.1% buffer (e.g. Sodium HydrogenPhosphate), about 1% protectant (e.g. Trehalose), and about 88.8% water.

In some embodiments, the present formulation is that of TABLE 7.

In some embodiments, the present formulation includes about 0.5-2.5%beta-lactamase, about 0.1-1% pore former, about 5-15% matrix, about0.1-1% lubricant, about 0.1-1% buffer, and about 80-90% water. In someembodiments, the present formulation includes about 0.5-2.5%beta-lactamase (e.g. P3A), about 0.1-1% pore former (e.g. HPMCAS-MF),about 5-15% matrix (e.g. Aquacoat (FMC)), about 0.1-1% lubricant (e.g.Sodium-Stearyl Fumarate), about 0.1-1% buffer (e.g. Sodium HydrogenPhosphate), and about 80-90% water.

In some embodiments, the present formulation includes about 2.5%beta-lactamase, about 0.5% pore former, about 10% matrix, about 0.5%lubricant, about 0.5% buffer, and about 90% water. In some embodiments,the present formulation includes about 2.5% beta-lactamase (e.g. P3A),about 0.5% pore former (e.g. HPMCAS-MF), about 10% matrix (e.g. Aquacoat(FMC)), about 0.5% lubricant (e.g. Sodium-Stearyl Fumarate), about 0.5%buffer (e.g. Sodium Hydrogen Phosphate), and about 90% water.

In some embodiments, the present formulation includes about 2.3%beta-lactamase, about 0.3% pore former, about 10% matrix, about 0.1%lubricant, about 0.1% buffer, and about 88.8% water. In someembodiments, the present formulation includes about 2.3% beta-lactamase(e.g. P3A), about 0.3% pore former (e.g. HPMCAS-MF), about 10% matrix(e.g. Aquacoat (FMC)), about 0.1% lubricant (e.g. Sodium-StearylFumarate), about 0.1% buffer (e.g. Sodium Hydrogen Phosphate), and about88.8% water.

In some embodiments, the present formulation is that of TABLE 8.

In various embodiments, the beta-lactamase described herein isformulated for microorganism-based release. In some embodiments, thebeta-lactamase is formulated for release by a genetically-modifiedmicroorganism, optionally selected from fungi, bacteria, and algae. Insome embodiments, the genetically-modified microorganism is resistant toone or more oral antibiotic. For example, the invention may pertain to agenetically-modified microorganism comprising one or morebeta-lactamases that is formulated for GI tract delivery as describedherein and that releases the beta-lactamases, e.g. by secretion. Forexample, a genetically-modified microorganism comprising one or morebeta-lactamases may be formulated for release in the distal smallintestine and/or colon and, when released, in turn, secretes orotherwise releases (e.g. via genetically-modified microorganism death ordigestion) the beta-lactamase so it may eliminate residual or excessoral antibiotic (e.g. active antibiotic that is not absorbed from the GItract after an oral dose or is returned in active form to the intestinaltract from the systemic circulation) and prevent GI tract microbiotadisruption.

In various embodiments, the genetically-modified microorganismcomprising one or more beta-lactamases is formulated so as to deliverviable recombinant yeast cells to the intestines where activebeta-lactamases are secreted by the genetically-modified microorganisms.In one embodiment, the genetically-modified microorganism comprising oneor more beta-lactamases is formulated as an enteric-coated capsule whichdirectly releases the recombinant genetically-modified microorganism inthe intestines. In other embodiments, the genetically-modifiedmicroorganism comprising one or more beta-lactamases can be formulatedas a gelatin capsule, or the genetically-modified microorganismcomprising one or more beta-lactamases can be dissolved in a liquid andingested. In such embodiments, the genetically-modified microorganismcomprising one or more beta-lactamases is delivered anywhere along theGI tract. As described herein, the genetically-modified microorganismcomprising one or more beta-lactamases can be released in the distalsmall intestine and/or the colon; however, delivery anywhere in the GItract is also imagined, for example, where the genetically-modifiedmicroorganism comprising one or more beta-lactamases is able to transitto the area of interest without loss of activity or disruption of thesystemic activity of the oral antibiotics. By way of illustration, insome embodiments, a recombinant yeast cell, for example, Saccharomycesboulardii, is resistant to stomach acid and remains viable duringtransit to the intestine, where it secretes active beta-lactamases forneutralizing residual or excess oral antibiotic (e.g. active antibioticthat is not absorbed from the GI tract after an oral dose or is returnedin active form to the intestinal tract from the systemic circulation) inthe lower GI tract.

In some embodiments, genetically-modified microorganism comprising oneor more beta-lactamases quickly transits through the small intestine buttransits slowly in the colon and therefore remains in the colon longerand any beta lactamase it secretes or releases concentrates in thecolon.

In some embodiments, the genetically-modified microorganism is a yeastcell. In various embodiments, the yeast cell is selected fromSaccharomyces spp., Hansenula spp., Kluyveromyces spp.Schizzosaccharomyces spp. Zygosaccharoinyces spp., Pichia spp., Monascusspp., Geotrichum spp. and Yarrowia spp. In various embodiments, thepresent invention contemplates expression of a beta-lactamase in arecombinant yeast cell. The recombinant yeast cell may be generated bystable integration into yeast chromosomal DNA of expression cassette(s)that encode and can express the one or more beta-lactamases.Alternatively, recombinant yeast cell may be generated using a processin which the yeast maintains an expression cassette(s) that encode andcan express the one or more beta-lactamases on a stable episome. Therecombinant yeast cell may be any yeast cell that is capable ofsurviving in the mammalian intestine. In various embodiments, the yeastcell has a known probiotic capacity, such as yeast strains selected fromkefir, kombucha or dairy products. I

In one embodiment, the recombinant yeast cell is Saccharomycescerevisiae. In another embodiment, the recombinant yeast cell is theSaccharomyces cerevisiae subspecies Saccharomyces boulardii (by way ofnon-limiting example, ATCC 74352 and/or any cells in U.S. Pat. Nos.6,010,695 and 7,799,328 the contents of which are hereby incorporated byreference in their entirety). S. cerevisiae has been marketed for over40 years as a probiotic. It has been used for the prevention and thetreatment of diarrheal diseases, including antibiotic-associateddiarrhea and C. difficile infection (reviewed by Kelesidis andPothoulakis, 2012; Hatoum et al., 2012). S. boulardii differs from otherS. cerevisiae strains as the optimal growth temperature of S. boulardiiis 37° C. while other strains prefer lower temperatures (between 30 and33° C.), S. boulardii is resistant to low pH and is highly tolerant tobile acids (Edwards-Ingram et al., 2007; Graff et al., 2008). S.boulardii was demonstrated to survive the intestinal tract in humans(Klein et al., 1993) where 0.1% viable yeast was recovered in fecesafter a single administration of 10¹⁰ cells. Concurrent antibiotictreatment increased recovery two-fold (Klein et al., 1993).

In some embodiments, the genetically-modified microorganism is abacterial cell. In some embodiments, the bacterial cell is a Bacillusspp. In some embodiments, the genetically-modified microorganism is analgal cell (e.g. Chlamydomonas spp., e.g. Chlamydomonas reinhardtii) orthe chloroplasts thereof.

In some embodiments, the genetically-modified microorganism is one ormore of Saccharomyces boulardii; Lactobacillus rhamnosus GG;Lactobacillus plantarum 299v; Clostridium butyricum M588; Clostridiumdifficile VP20621 (non-toxigenic C. difficile strain); combination ofLactobacillus casei, Lactobacillus acidophilus (Bio-K+CL1285);combination of Lactobacillus casei, Lactobacillus bulgaricus,Streptococcus thermophilus (Actimel); combination.of Lactobacillusacidophilus, Bifidobacterium bifidum (Florajen3); combination ofLactobacillus acidophilus, Lactobacillus bulgaricus delbrueckii subsp.bulgaricus, Lactobacillus bulgaricus casei, Lactobacillus bulgaricusplantarum, Bifidobacterium longum, Bifidobacterium infantis,Bifidobacterium breve, and Streptococcus salivarius subsp.thermophilus(VSL#3)).

Such genetically-modified microorganisms may be administered asdescribed herein, including by way of example, enterally, such asorally.

Administration and Dosage

It will be appreciated that the actual dose of the beta-lactamase(and/or additional therapeutic agents) to be administered according tothe present invention will vary according to, for example, theparticular dosage form and the mode of administration. Many factors thatmay modify the action of the beta-lactamase (e.g., body weight, gender,diet, time of administration, route of administration, rate ofexcretion, condition of the subject, drug combinations, geneticdisposition and reaction sensitivities) can be taken into account bythose skilled in the art. Administration can be carried out continuouslyor in one or more discrete doses within the maximum tolerated dose.Optimal administration rates for a given set of conditions can beascertained by those skilled in the art using conventional dosageadministration tests.

Individual doses of the beta-lactamase (and/or additional therapeuticagents) can be administered in unit dosage forms (e.g., tablets orcapsules) containing, for example, from about 0.01 mg to about 5,000 mg,from about 0.01 mg to about 4,000 mg, from about 0.01 mg to about 3,000mg, from about 0.01 mg to about 2,000 mg, from about 0.01 mg to about1,000 mg, from about 0.01 mg to about 950 mg, from about 0.01 mg toabout 900 mg, from about 0.01 mg to about 850 mg, from about 0.01 mg toabout 800 mg, from about 0.01 mg to about 750 mg, from about 0.01 mg toabout 700 mg, from about 0.01 mg to about 650 mg, from about 0.01 mg toabout 600 mg, from about 0.01 mg to about 550 mg, from about 0.01 mg toabout 500 mg, from about 0.01 mg to about 450 mg, from about 0.01 mg toabout 400 mg, from about 0.01 mg to about 350 mg, from about 0.01 mg toabout 300 mg, from about 0.01 mg to about 250 mg, from about 0.01 mg toabout 200 mg, from about 0.01 mg to about 150 mg, from about 0.01 mg toabout 100 mg, from about 0.1 mg to about 90 mg, from about 0.1 mg toabout 80 mg, from about 0.1 mg to about 70 mg, from about 0.1 mg toabout 60 mg, from about 0.1 mg to about 50 mg, from about 0.1 mg toabout 40 mg, from about 0.1 mg to about 30 mg, from about 0.1 mg toabout 20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg toabout 5 mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about1 mg of the active ingredient per unit dosage form, or from about 5 mgto about 80 mg per unit dosage form. For example, a unit dosage form caninclude about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg,about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg,about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg,about 8 mg, about 9 mg about 10 mg, about 15 mg, about 20 mg, about 25mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg,about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 150mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900mg, about 950 mg, about 1,000 mg, about 2,000 mg, about 3,000 mg, about4,000 mg, or about 5,000 mg of the active ingredient, inclusive of allvalues and ranges therebetween.

In one embodiment, the beta-lactamase (and/or additional therapeuticagents) is administered at an amount of from about 0.01 mg to about 100mg daily, an amount of from about 0.01 mg to about 5,000 mg daily, about0.01 mg to about 4,000 mg daily, about 0.01 mg to about 3,000 mg daily,about 0.01 mg to about 2,000 mg daily, about 0.01 mg to about 1,000 mgdaily, from about 0.01 mg to about 950 mg daily, from about 0.01 mg toabout 900 mg daily, from about 0.01 mg to about 850 mg daily, from about0.01 mg to about 800 mg daily, from about 0.01 mg to about 750 mg daily,from about 0.01 mg to about 700 mg daily, from about 0.01 mg to about650 mg daily, from about 0.01 mg to about 600 mg daily, from about 0.01mg to about 550 mg daily, from about 0.01 mg to about 500 mg daily, fromabout 0.01 mg to about 450 mg daily, from about 0.01 mg to about 400 mgdaily, from about 0.01 mg to about 350 mg daily, from about 0.01 mg toabout 300 mg daily, from about 0.01 mg to about 250 mg daily, from about0.01 mg to about 200 mg daily, from about 0.01 mg to about 150 mg daily,from about 0.1 mg to about 100 mg daily, from about 0.1 mg to about 95mg daily, from about 0.1 mg to about 90 mg daily, from about 0.1 mg toabout 85 mg daily, from about 0.1 mg to about 80 mg daily, from about0.1 mg to about 75 mg daily, from about 0.1 mg to about 70 mg daily,from about 0.1 mg to about 65 mg daily, from about 0.1 mg to about 60 mgdaily, from about 0.1 mg to about 55 mg daily, from about 0.1 mg toabout 50 mg daily, from about 0.1 mg to about 45 mg daily, from about0.1 mg to about 40 mg daily, from about 0.1 mg to about 35 mg daily,from about 0.1 mg to about 30 mg daily, from about 0.1 mg to about 25 mgdaily, from about 0.1 mg to about 20 mg daily, from about 0.1 mg toabout 15 mg daily, from about 0.1 mg to about 10 mg daily, from about0.1 mg to about 5 mg daily, from about 0.1 mg to about 3 mg daily, fromabout 0.1 mg to about 1 mg daily, or from about 5 mg to about 80 mgdaily. In various embodiments, the beta-lactamase (and/or additionaltherapeutic agents) is administered at a daily dose of about 0.01 mg,about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg,about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mgabout 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg,about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about90 mg, about 95 mg, about 100 mg, about 150 mg, about 200 mg, about 250mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1,000mg, about 2,000 mg, about 3,000 mg, about 4,000 mg, or about 5,000 mginclusive of all values and ranges therebetween.

In some embodiments, a suitable dosage of the beta-lactamase (and/oradditional therapeutic agents) is in a range of about 0.01 mg/kg toabout 100 mg/kg of body weight of the subject, for example, about 0.01mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg,about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg,about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, about 20 mg/kg bodyweight, about 30 mg/kg body weight, about 40 mg/kg body weight, about 50mg/kg body weight, about 60 mg/kg body weight, about 70 mg/kg bodyweight, about 80 mg/kg body weight, about 90 mg/kg body weight, or about100 mg/kg body weight, inclusive of all values and ranges therebetween.In other embodiments, a suitable dosage of the beta-lactamases (and/oradditional therapeutic agents) in a range of about 0.01 mg/kg to about100 mg/kg of body weight, in a range of about 0.01 mg/kg to about 90mg/kg of body weight, in a range of about 0.01 mg/kg to about 80 mg/kgof body weight, in a range of about 0.01 mg/kg to about 70 mg/kg of bodyweight, in a range of about 0.01 mg/kg to about 60 mg/kg of body weight,in a range of about 0.01 mg/kg to about 50 mg/kg of body weight, in arange of about 0.01 mg/kg to about 40 mg/kg of body weight, in a rangeof about 0.01 mg/kg to about 30 mg/kg of body weight, in a range ofabout 0.01 mg/kg to about 20 mg/kg of body weight, in a range of about0.01 mg/kg to about 10 mg/kg of body weight, in a range of about 0.01mg/kg to about 9 mg/kg of body weight, in a range of about 0.01 mg/kg toabout 8 mg/kg of body weight, in a range of about 0.01 mg/kg to about 7mg/kg of body weight, in a range of 0.01 mg/kg to about 6 mg/kg of bodyweight, in a range of about 0.05 mg/kg to about 5 mg/kg of body weight,in a range of about 0.05 mg/kg to about 4 mg/kg of body weight, in arange of about 0.05 mg/kg to about 3 mg/kg of body weight, in a range ofabout 0.05 mg/kg to about 2 mg/kg of body weight, in a range of about0.05 mg/kg to about 1.5 mg/kg of body weight, or in a range of about0.05 mg/kg to about 1 mg/kg of body weight.

In accordance with certain embodiments of the invention, thebeta-lactamase may be administered, for example, more than once daily,about once per day, about every other day, about every third day, aboutonce a week, about once every two weeks, about once every month, aboutonce every two months, about once every three months, about once everysix months, or about once every year.

Additional Therapeutic Agents and Combination Therapy or Co-Formulation

Administration of the present formulations may be combined withadditional therapeutic agents. Co-administration of the additionaltherapeutic agent and, the present formulations may be simultaneous orsequential. Further, the present formulations may comprise an additionaltherapeutic agent (e.g. via co-formulation). For example, the additionaltherapeutic agent and the beta-lactamase may be combined into a singleformulation.

In one embodiment, the additional therapeutic agent and thebeta-lactamase are administered to a subject simultaneously. The term“simultaneously” as used herein, means that the additional therapeuticagent and the beta-lactamase are administered with a time separation ofno more than about 60 minutes, such as no more than about 30 minutes, nomore than about 20 minutes, no more than about 10 minutes, no more thanabout 5 minutes, or no more than about 1 minute. Administration of theadditional therapeutic agent and the beta-lactamase can be bysimultaneous administration of a single formulation (e.g., a formulationcomprising the additional therapeutic agent and the beta-lactamase) orof separate formulations (e.g., a first formulation including theadditional therapeutic agent and a second formulation including thebeta-lactamase).

Co-administration does not require the additional therapeutic agents tobe administered simultaneously, if the timing of their administration issuch that the pharmacological activities of the additional therapeuticagent and the beta-lactamase overlap in time. For example, theadditional therapeutic agent and the beta-lactamase can be administeredsequentially. The term “sequentially” as used herein means that theadditional therapeutic agent and the beta-lactamase are administeredwith a time separation of more than about 60 minutes. For example, thetime between the sequential administration of the additional therapeuticagent and the beta-lactamase can be more than about 60 minutes, morethan about 2 hours, more than about 5 hours, more than about 10 hours,more than about 1 day, more than about 2 days, more than about 3 days,or more than about 1 week apart. The optimal administration times willdepend on the rates of metabolism, excretion, and/or the pharmacodynamicactivity of the additional therapeutic agent and the beta-lactamasebeing administered. Either the additional therapeutic agent or thebeta-lactamase may be administered first.

In a further embodiment, the additional therapeutic agent and thebeta-lactamase are administered to a subject simultaneously but therelease of additional therapeutic agent and the beta-lactamase fromtheir respective dosage forms (or single unit dosage form ifco-formulated) in the GI tract occurs sequentially.

Co-administration also does not require the additional therapeuticagents to be administered to the subject by the same route ofadministration. Rather, each additional therapeutic agent can beadministered by any appropriate route, for example, parenterally ornon-parenterally.

In some embodiments, the additional therapeutic agent is ananti-bacterial agent, which includes, but is not limited to,cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil,cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, andceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin,tequin, avelox, and norflox); tetracycline antibiotics (tetracycline,minocycline, oxytetracycline, and doxycycline); penicillin antibiotics(amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin,vancomycin, and methicillin); monobactam antibiotics (aztreonam); andcarbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, andmeropenem). In some embodiments, any of the penicillins andcephalosporins described herein may be the additional therapeutic agent.

In some embodiments, the additional therapeutic agent is abeta-lactamase inhibitor. Exemplary beta-lactamase inhibitors include,for example, tazobactam, sulbactam, clavulanic acid, avibactam,monobactam derivatives, ATMO derivatives, penems (e.g., BRL42715 andderivatives thereof, Syn1012, oxapenems, trinems,1-β-methylcarbapenems), penicillin and cephalosporin sulfone derivatives(e.g., C-21C-3-substituted penicillin and cephalosporin sulfones,C-6-substituted penicillin sulfones), non-β-lactam inhibitors (e.g.,boronic acid transition state analogs, phophonates, NXL104, hydroxmates)and metallo-β-lactamase inhibitors such as thiol derivatives, pyridinedicarboxylates, trifluoromethyl ketones and alcohols, carbapenemanalogs, tricyclic natural products, succinate derivatives, andC-6-mercaptomethyl penicillinates.

In some embodiments, the additional therapeutic agent is an adjunctivetherapy that is used in, for example, the treatment of CDI as describedherein. In some embodiments, the additional therapeutic agent ismetronidazole (e.g. FLAGYL), fidaxomicin (e.g. DIFICID), or vancomycin(e.g. Vancocin), rifaximin, charcoal-based binders/adsorbents (e.g.DAV132), fecal bacteriotherapy, probiotic therapy (see, e.g., Intnat'l JInf Dis, 16 (11): e786, the contents of which are hereby incorporated byreference, illustrative probiotics include Saccharomyces boulardii;Lactobacillus rhamnosus GG; Lactobacillus plantarum 299v; Clostridiumbutyricum M588; Clostridium difficile VP20621 (non-toxigenic C.difficile strain); combination of Lactobacillus casei, Lactobacillusacidophilus (Bio-K+CL1285); combination of Lactobacillus casei,Lactobacillus bulgaricus, Streptococcus thermophilus (Actimel);combination of Lactobacillus acidophilus, Bifidobacterium bifidum(Florajen3); combination of Lactobacillus acidophilus, Lactobacillusbulgaricus delbrueckii subsp. bulgaricus, Lactobacillus bulgaricuscasei, Lactobacillus bulgaricus plantarum, Bifidobacterium longum,Bifidobacterium infantis, Bifidobacterium breve, Streptococcussalivarius subsp.thermophilus (VSL#3)) and antibody or other biologictherapy (e.g. monoclonal antibodies against C. difficile toxins A and Bas described in N Engl J Med. 2010; 362(3):197, the contents of whichare hereby incorporated by reference in their entirety; neutralizingbinding proteins, for example, arranged as multimers, which are directedto one or more of SEQ ID NOs. recited in United States PatentPublication No. 2013/0058962 (e.g. one or more of SEQ ID Nos.: 59, 60,95, 67, 68, and 87), the contents of which are hereby incorporated byreference); or any neutralizing binding protein directed against C.difficile binary toxin.

In some embodiments, the additional therapeutic agent is anantidiarrheal agent. Antidiarrheal agents suitable for use in thepresent invention include, but are not limited to, DPP-IV inhibitors,natural opioids, such as tincture of opium, paregoric, and codeine,synthetic opioids, such as diphenoxylate, difenoxin and loperamide,bismuth subsalicylate, lanreotide, vapreotide and octreotide, motilnantagonists, COX2 inhibitors like celecoxib, glutamine, thalidomide andtraditional antidiarrheal remedies, such as kaolin, pectin, berberineand muscarinic agents.

In some embodiments, the additional therapeutic agent is ananti-inflammatory agent such as steroidal anti-inflammatory agents ornon-steroidal anti-inflammatory agents (NSAIDS). Steroids, particularlythe adrenal corticosteroids and their synthetic analogues, are wellknown in the art. Examples of corticosteroids useful in the presentinvention include, without limitation, hydroxyltriamcinolone,alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasonedipropionate, betamethasone, benzoate, betamethasone dipropionate,betamethasone valerate, clobetasol valerate, desonide, desoxymethasone,dexamethasone, diflorasone diacetate, diflucortolone valerate,fluadrenolone, fluclorolone acetonide, flumethasone pivalate,fluosinolone acetonide, fluocinonide, flucortine butylester,fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone,halcinonide, hydrocortisone acetate, hydrocortisone butyrate,methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone,flucetonide, fludrocortisone, difluorosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, clocortelone, clescinolone,dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone,fluperolone, fluprednisolone, hydrocortisone, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate.(NSAIDS) that may be used in the present invention, include but are notlimited to, salicylic acid, acetyl salicylic acid, methyl salicylate,glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid,ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone,and indomethacin. Additional anti-inflammatory agents are described, forexample, in U.S. Pat. No. 4,537,776, the entire contents of which isincorporated by reference herein.

In some embodiments, the additional therapeutic agent may be ananalgesic. Analgesics useful in the compositions and methods of thepresent invention include, without limitation, morphine, codeine,heroine, methadone and related compounds, thebaine, orpiavine, and theirderivatives, buprenorphine, the piperidines, morphinans, benzomorphans,tetrahydroisoquinolines, thiambutanes, benzylamines, tilidine, viminol,nefopam, capsaicin(8-methyl-N-vanillyl-6E-nonenamide), “synthetic”capsaicin(N-vanillylnonamide), and related compounds.

In some embodiments, the additional therapeutic agent may be ananti-viral agent that includes, but is not limited to, Abacavir,Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir,Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir,Emtricitabine, Enfuvirtibe, Etravirine, Famciclovir, and Foscarnet.

For all additional therapeutic agent compositions and methods, targetingto various parts of the GI tract may be employed as described herein.

In some embodiments, the present formulations are administered to asubject to avoid treatment with an additional therapeutic agent. Forexample, in the context of preventing C. difficile infection (CDI)and/or a C. difficile-associated disease, the present formulations maybe provided to a subject to avoid the necessity of receiving, forexample, vancomycin.

Methods of Treatment

In various aspects, the present invention provides methods forprotecting a subject's gastrointestinal microbiome, comprisingadministering an effective amount of a pharmaceutical compositioncomprising a beta-lactamase, for example, any of the formulationsdescribed herein, to a subject who is undergoing treatment or hasrecently undergone treatment with an oral antibiotic. The beta-lactamaseis capable of deactivating (by way of non-limitation, hydrolyzing) theoral antibiotic. In various embodiments, the oral antibiotic is one ormore of a ceftriaxone, cefotaxime, cefazolin, cefoperazone, cefuroxime,and piperacillin.

In various embodiments, the subjects include, but are not limited to,subjects that are at a particular risk for a microbiome-mediateddisorder, such as those undergoing treatment or has recently undergonetreatment with an oral antibiotic. For example, the subject may betaking an oral antibiotic during the past 30 or so days and/or have animmune system that is weak (e.g. from a chronic illness) and/or arewomen and/or are elderly (e.g. over about 65 years old) and/or areelderly woman and/or undergo treatment with for heartburn or stomachacid disorders (e.g. with agents such as PREVACID, TAGAMET, PRILOSEC, orNEXIUM and related drugs) and/or have recently been in the hospital,including in an intensive care unit, or live in a nursing home.Accordingly, in some embodiments, the methods and uses of the presentinvention treat or prevent a nosocomial infection and/or a secondaryemergent infection and/or a hospital acquired infection (HAI).

In some embodiments, the methods and uses of the present inventioninclude those in which an initial and/or adjunctive therapy isadministered to a subject. Initial and/or adjunctive therapy indicatestherapy that is used to treat for example, a microbiome-mediateddisorder or disease upon detection of such disorder or disease. In someembodiments, the initial and/or adjunctive therapy is one or more ofmetronidazole, vancomycin, fidaxomicin, rifaximin, charcoal-basedbinder/adsorbent, fecal bacteriotherapy, probiotic therapy, and antibodytherapy, as described herein. In various embodiments, the methods anduses of the present invention include use of the pharmaceuticalcompositions and formulations including beta-lactamase (and/oradditional therapeutic agent) as an adjuvant to any of these initialand/or adjunctive therapies (including co-administration or sequentialadministration). In various embodiments, the methods and uses of thepresent invention include use of the pharmaceutical compositions andformulations including beta-lactamase (and/or additional therapeuticagent) in a subject undergoing initial and/or adjunctive therapies.

In some embodiments, the methods and uses of the present inventioninclude those in which an oral antibiotic and a beta-lactamase inhibitorare administered to a subject. In various embodiments, the subject maybe receiving a co-formulation of an oral antibiotic with one or morebeta-lactamase inhibitors (e.g. Augmentin is a mixture of amoxicillinand clavulanic acid). Such co-formulations include, but are not limitedto, amoxicillin-clavulanic acid (Augmentin, ticarcillin-clavulanic acid(Timentin), ampicillin-sulbactam (Sultamicillin, e.g. Unasyn),piperacillin-tazobactam (Zosyn), and cefoperazone-sulbactam. In variousembodiments, methods of the present invention comprise furtheradministering a beta-lactamase inhibitor that releases in the GI tractproximal to the beta-lactamase. In an embodiment, the beta-lactamaseinhibitor may be released at various parts of the GI tract where theoral antibiotic may be active. For example, the beta-lactamase inhibitormay be released at the stomach, duodenum, jejunum and ileum. Exemplarybeta-lactamase inhibitors include, for example, tazobactam, sulbactam,clavulanic acid, avibactam, monobactam derivatives, ATMO derivatives,penems (e.g., BRL42715 and derivatives thereof, Syn1012, oxapenems,trinems, 1-β-methylcarbapenems), penicillin and cephalosporin sulfonederivatives (e.g., C-2/C-3-substituted penicillin and cephalosporinsulfones, C-6-substituted penicillin sulfones), non-β-lactam inhibitors(e.g., boronic acid transition state analogs, phophonates, NXL104,hydroxmates) and metallo-β-lactamase inhibitors such as thiolderivatives, pyridine dicarboxylates, trifluoromethyl ketones andalcohols, carbapenem analogs, tricyclic natural products, succinatederivatives, and C-6-mercaptomethyl penicillinates.

In various embodiments, the methods of the invention comprise treatingor preventing a microbiome-mediated disorder. Illustrativemicrobiome-mediated disorder includes, but are not limited to, forexample, those found in Table 3 of WO2014/121298, the entire contents ofwhich are incorporated herein by reference. For example, themicrobiome-mediated disorder may be selected from an antibiotic-inducedadverse effect, a C. difficile infection (CDI), a C.difficile-associated disease, ulcerative colitis, Crohn's disease, andirritable bowel syndrome. In various embodiments, themicrobiome-mediated disorder is an antibiotic-induced adverse effect, aC. difficile infection (CDI), or a C. difficile-associated disease. Inan embodiment, the present invention provides methods for treating anantibiotic-induced adverse effect in the GI tract, comprisingadministering an effective amount of a pharmaceutical composition orformulation including beta-lactamase (and/or additional therapeuticagent) described herein to a subject who is undergoing treatment or hasrecently undergone treatment with an oral antibiotic. In anotherembodiment, the present invention provides methods for preventing anantibiotic-induced adverse effect in the GI tract, comprisingadministering an effective amount of a pharmaceutical composition orformulation including beta-lactamase (and/or additional therapeuticagent) described herein to a subject who is undergoing treatment or hasrecently undergone treatment with an oral antibiotic.

In an embodiment, the present invention provides methods for treating C.difficile infection (CDI) and/or a C. difficile-associated disease,comprising administering an effective amount of a pharmaceuticalcomposition or formulation including beta-lactamase (and/or additionaltherapeutic agent) described herein to a subject who is undergoingtreatment or has recently undergone treatment with an oral antibiotic.In another embodiment, the present invention provides methods forpreventing C. difficile infection (CDI) and/or a C. difficile-associateddisease, comprising administering an effective amount of apharmaceutical composition or formulation including beta-lactamase(and/or additional therapeutic agent) described herein to a subject whois undergoing treatment or has recently undergone treatment with an oralantibiotic.

In various embodiments, the present invention relates to methods ofpreventing and/or reducing the likelihood that a subject becomesafflicted with an antibiotic-associated adverse effect (e.g. Clostridiumdifficile infection, antibiotic associated diarrhea) by administering aneffective amount of a beta-lactamase formulation as described herein,such as those presented in TABLES 1, 5, 7, and 8. In some embodiments,the formulation, optionally, in the form of a capsule (e.g., a hardgelatin or HPMC capsule) comprises a plurality of enteric-coatedbeta-lactamase-containing pellets. In some embodiments, the formulation,optionally, in the form of a capsule (e.g., a hard gelatin or HPMCcapsule) comprises a plurality of beta-lactamase-containing pelletscoated with an osmotic rupture coating.

In some embodiments, the beta-lactamase-containing pellets (or eachindividual pellet) comprises a beta-lactamase (e.g. P3A, or the otherbeta-lactamase agents described herein, and variants thereof), a sucrosesphere, which the beta-lactamase, for example, P3A or a variant, issprayed onto, a binder excipient (e.g., hydroxypropylcellulose (HPC)),an enteric polymer (e.g., EUDRAGIT L 30 D-55), a plasticizer (e.g.,triethyl citrate), a glidant (e.g., glyceryl monostearate), anemulsifier, and buffer salts, where to subject is receiving an oralbeta-lactam antibiotic which is a substrate of the beta-lactamase. Invarious embodiments, the above method involves a formulation, optionallyin the form of a capsule e.g., a hard gelatin or HPMC capsule)comprising a plurality of enteric-coated beta-lactamase-containingpellets, the pellets (or each individual pellet) comprising about 10-20%by weight of beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof). For example, thebeta-lactamase (e.g. P3A, or the other beta-lactamase agents describedherein, and variants thereof) may be present at about 10%, about 11%,about 12%, about 13%, about-14%, about 15%, about 16%, about 17%, about18%, about 19%, or about 20% by weight. In some embodiments, the pellets(or each individual pellet) comprise about 20-30% by weight sucrosesphere, which the beta-lactamase, for example, P3A or a variant, issprayed onto. For example, the sucrose sphere may be present at about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, or about 30% by weight. In variousembodiments, the pellets (or each individual pellet) comprise about30-40% by weight a binder excipient (e.g., hydroxypropylcellulose(HPC)). For example, the binder excipient may be present at about 30%,about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about37%, about 38%, about 39%, or about 40% by weight. In some embodiments,the pellets (or each individual pellet) comprise about 15-25% by weightan enteric polymer (e.g., EUDRAGIT L 30 D-55). For example, the entericpolymer may be present at about 15%, about 16%, about 17%, about 18%,about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, orabout 25% by weight. In some embodiments, the pellets (or eachindividual pellet) comprise about 1.5-2.5% by weight of plasticizer(e.g., triethyl citrate). For example, the plasticizer may be present atabout 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%,about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5% by weight. Insome embodiments, the pellets (or each individual pellet) comprise about0.5-1.5% by weight glidant (e.g., glyceryl monostearate). For example,the glidant may be present at about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about1.4%, or about 1.5% by weight. In some embodiments, the pellets (or eachindividual pellet) comprise about 0.1-1.0% by weight emulsifier (e.g.polysorbate-80). For example, the emulsifier may be present at about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, or about 1% by weight. In someembodiments, the pellets (or each individual pellet) further compriseabout 1-2% by weight buffer salts. For example, the buffer salts may bepresent at about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%,about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% by weight.The weight as described herein refers to the total weight of allcomponents excluding the weight of the capsule itself. In variousembodiments, the above method involves a formulation, optionally in theform of a pellet (or each individual pellet) comprising about 16% byweight of the beta-lactamase (e.g. P3A, or the other beta-lactamaseagents described herein, and variants thereof); about 23% by weightsucrose sphere; about 35% by weight a binder excipient (e.g.,hydroxypropylcellulose (HPC)); about 21% by weight an enteric polymer(e.g., EUDRAGIT L 30 D-55); about 2% by weight of plasticizer (e.g.,triethyl citrate); about 1% by weight glidant (e.g., glycerylmonostearate); about 0.5% by weight emulsifier (e.g. polysorbate-80);and about 2% by weight buffer salts. The weight as described hereinrefers to the total weight of all components excluding the weight of thecapsule itself. For example, the pellets (or each individual pellet)comprise about 15.8% by weight of the beta-lactamase (e.g. P3A, or theother beta-lactamase agents described herein, and variants thereof);about 23.3% by weight sucrose sphere; about 35% by weight a binderexcipient (e.g., hydroxypropylcellulose (HPC)); about 20.8% by weight anenteric polymer (e.g., EUDRAGIT L 30 D-55); about 2.1% by weight ofplasticizer (e.g., triethyl citrate); about 1.0% by weight glidant(e.g., glyceryl monostearate); about 0.4% by weight emulsifier (e.g.polysorbate-80); and about 1.6% by weight buffer salts. The weight asdescribed herein refers to the total weight of all components excludingthe weight of the capsule itself.

In various embodiments, the antibiotic-induced adverse effect and/or CDIor C. difficile-associated disease is one or more of:antibiotic-associated diarrhea, C. difficile diarrhea (CDD), C.difficile intestinal inflammatory disease, colitis, pseudomembranouscolitis, fever, abdominal pain, dehydration and disturbances inelectrolytes, megacolon, peritonitis, and perforation and/or rupture ofthe colon. Additional diseases, disorders and conditions which aresuitable for treatment with the compositions and methods of theinvention include those listed in Table 3 of WO2014/121298, the entirecontents of which are incorporated herein by reference.

In various embodiments, the present uses and methods pertain toco-treatment (simultaneously or sequentially) with the pharmaceuticalcomposition or formulation including beta-lactamase (and/or additionaltherapeutic agent) described herein and/or any initial and/or adjunctivetherapy, or treatment with a co-formulation of the pharmaceuticalcomposition or formulation including beta-lactamase (and/or anyadditional therapeutic agent) described herein and/or any initial and/oradjunctive therapy for treatment of the various diseases describedherein.

In various embodiments, the microbiome-mediated disorder is treated orprevented in the context of initial onset or relapse/recurrence (e.g.due to continued or restarted antibiotic therapy). For example, in asubject that has previously suffered from a microbiome-mediated disorder(e.g., CDI), the present pharmaceutical composition or formulationincluding beta-lactamase (and/or additional therapeutic agent) may beadministered upon the first symptoms of recurrence in the subject. Byway of non-limiting example, symptoms of recurrence include, in a mildcase, about 5 to about 10 watery bowel movements per day, no significantfever, and only mild abdominal cramps while blood tests may show a mildrise in the white blood cell count up to about 15,000 (normal levels areup to about 10,000), and, in a severe case, more than about 10 waterystools per day, nausea, vomiting, high fever (e.g. about 102-104° F.),rectal bleeding, severe abdominal pain (e.g. with tenderness), abdominaldistention, and a high white blood count (e.g. of about 15,000 to about40,000).

Regardless of initial onset or relapse/recurrence, themicrobiome-mediated disorder may be diagnosed via any of the symptomsdescribed herein (e.g. watery diarrhea about 3 or more times a day forabout 2 days or more, mild to bad cramping and pain in the belly, fever,blood or pus in the stool, nausea, dehydration, loss of appetite, lossof weight, etc.). Regardless of initial onset or relapse/recurrence, themicrobiome-mediated disorder may also be diagnosed via enzymeimmunoassays (e.g. to detect the C. difficile toxin A or B antigenand/or glutamine dehydrogenase (GDH), which is produced by C. difficileorganisms), polymerase chain reactions (e.g., to detect the C. difficiletoxin A or B gene or a portion thereof (e.g. tcdA or tcdB), includingthe ILLUMIGENE LAMP assay), a cell cytotoxicity assay. For example, anyof the following tests may be used: Meridian ImmunoCard Toxins A/B;Wampole Toxin A/B Quik Chek; Wampole C. diff Quik Chek Complete; RemelXpect Clostridium difficile Toxin A/B; Meridian Premier Toxins A/B;Wampole C. difficile Tox A/B II; Remel Prospect Toxin A/B EIA;Biomerieux Vidas C. difficile Toxin A&B; BD Geneohm C. diff; ProdesseProgastro CD; and Cepheld Xpert C. diff. In various embodiments, theclinical sample is a subject's stool sample.

Also a flexible sigmoidoscopy “scope” test and/or an abdominal X-rayand/or a computerized tomography (CT) scan, which provides images ofyour colon, may be used in assessing a subject (e.g. looking forcharacteristic creamy white or yellow plaques adherent to the wall ofthe colon). Further, biopsies (e.g. of any region of the GI tract) maybe used to assess a potential microbiome-mediated disorder (e.g., CDIand/or C. difficile associated disease) in subject.

In various embodiments, the methods and uses of the present inventionrelate to pharmaceutical compositions and formulations includingbeta-lactamase (and/or additional therapeutic agent) which release thebeta-lactamase (and/or additional therapeutic agent) in a location inthe GI tract in which it deactivates excess oral antibiotic residue. Inan embodiment, the methods and uses of the present invention relate topharmaceutical compositions and formulations including beta-lactamase(and/or additional therapeutic agent) which deactivate excess oralantibiotic residue before it enters the GI tract, including the smalland/or large intestine. In an embodiment, the methods and uses of thepresent invention relate to pharmaceutical compositions and formulationsincluding beta-lactamase (and/or additional therapeutic agent) whichdeactivate excess oral antibiotic residue before it enters the largeintestine. In an embodiment, the methods and uses of the presentinvention relate to pharmaceutical compositions and formulationsincluding beta-lactamase (and/or additional therapeutic agent) whichdeactivate excess oral antibiotic residue in the GI tract. In variousembodiments, the pharmaceutical compositions and formulations includingbeta-lactamase (and/or additional therapeutic agent) as described hereinreleases the beta-lactamase (and/or additional therapeutic agent) in alocation in the GI tract that is distal to the release of the oralantibiotic. In various embodiments, the beta-lactamase (and/oradditional therapeutic agent) is released in a location in the GI tractwhere it prevents a microbicidal activity of the residual or excess oralantibiotic (e.g. active antibiotic that is not absorbed from the GItract after an oral dose or is returned in active form to the intestinaltract from the systemic circulation) on GI tract microbiota.

In some embodiments, methods and uses of the present invention relate topharmaceutical compositions and formulation including beta-lactamase(and/or additional therapeutic agent) which maintain a normal intestinalmicrobiota and/or prevent the overgrowth of one or more pathogenicmicroorganisms in the GI tract of a subject.

In various embodiments, the present invention provides forpharmaceutical compositions and methods that mitigate or prevent theovergrowth of various coliforms in a subject's gut (including coliformsthat are virulent and/or antibiotic resistant). In various aspects, themethods, pharmaceutical compositions and formulations described hereinprevent or diminish secondary infections with resistant organisms andmay, in some embodiments, diminish beta-lactam resistance development.Further, the methods, pharmaceutical compositions and formulationsdescribed herein may allow for use of beta-lactam antibiotics which arecurrently avoided due to resistance concerns and/or reduce the need forco-administration or co-formulation with one or more beta-lactamaseinhibitors (e.g. Augmentin, Sultamicillin).

In various embodiments, the beta-lactamases and/or pharmaceuticalcompositions (and/or additional therapeutic agents) do not substantiallyinterfere with blood or plasma levels of an oral antibiotic. Forexample, the beta-lactamases and/or pharmaceutical compositions (and/oradditional therapeutic agents) of the present invention allow for asubject to receive an oral antibiotic that might be required for aninfection and do not interfere with the systemic activity of the oralantibiotic or the time above minimum inhibitory concentrations of theantibiotic in the plasma. In an embodiment, the beta-lactamases and/orpharmaceutical compositions (and/or additional therapeutic agents) doesnot substantially interfere with blood or plasma levels of the oralantibiotic. Rather, the beta-lactamases and/or pharmaceuticalcompositions (and/or additional therapeutic agents) inactivate residualor excess oral antibiotic (e.g. active antibiotic that is not absorbedfrom the GI tract after an oral dose or is returned in active form tothe intestinal tract from the systemic circulation) that may populateparts of the GI tract and in doing so, prevent the disruption of themicrobiota that is linked to the various disease states describedherein.

In various embodiments, the pharmaceutical compositions and formulationsincluding beta-lactamase and/or additional therapeutic agent are notsystemically absorbed. In some embodiments, the compositions andformulations including beta-lactamase (and/or additional therapeuticagent) do not interfere with the antibiotic absorption from the gutand/or or antibiotic enterohepatic recirculation enough to be clinicallyimportant.

In various embodiments, the pharmaceutical compositions and formulationsincluding beta-lactamase and/or additional therapeutic agent are used asan adjuvant for the treatment of H. pylori infection, e.g. in thegastric mucosa. For instance, the present pharmaceutical compositionsand formulations may be used as adjuvant to amoxicillin treatments (e.g.as an adjuvant to “triple therapy” (e.g. proton pump inhibitors such asomeprazole, pantoprazole, or rabeprazole and the antibioticsclarithromycin and amoxicillin, or metronidazole)). By way of example,the amoxicillin would be administered such that it is delivered to thestomach where it has a therapeutic effect and then it is deactivatedupon exiting the stomach by the pharmaceutical compositions andformulations (e.g. duodenally-released). Accordingly, provided hereinare methods of treating or preventing H. pylori infection in a subject'sstomach by administering a pharmaceutical compositions and formulationsincluding beta-lactamase and/or additional therapeutic agent describedherein. Further, the present methods are useful in treating orpreventing an H. pylori infection-related disease (by way ofnon-limiting example: ulcers (e.g. duodenal ulcers, peptic ulcerdisease), cancers (e.g. stomach cancer, gastric MALT lymphoma), anddyspepsia). In some of these embodiments, there is no requirement topreserve a systemic level of oral antibiotic.

In some embodiments, the terms “patient” and “subject” are usedinterchangeably. In some embodiments, the subject and/or animal is amammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow,pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee,or baboon. In other embodiments, the subject and/or animal is anon-mammal, such, for example, a zebrafish. In some embodiments, thesubject and/or animal may comprise fluorescently-tagged cells (with e.g.GFP). In some embodiments, the subject and/or animal is a transgenicanimal comprising a fluorescent cell.

In various embodiments, methods of the invention are useful in treatmenta human subject. In some embodiments, the human is a pediatric human. Inother embodiments, the human is an adult human. In other embodiments,the human is a geriatric human. In other embodiments, the human may bereferred to as a subject. In some embodiments, the human is a female. Insome embodiments, the human is a male.

In certain embodiments, the human has an age in a range of from about 1to about 18 months old, from about 18 to about 36 months old, from about1 to about 5 years old, from about 5 to about 10.years old, from about10 to about 15 years old, from about 15 to about 20 years old, fromabout 20 to about 25 years old, from about 25 to about 30 years old,from about 30 to about 35 years old, from about 35 to about 40 yearsold, from about 40 to about 45 years old, from about 45 to about 50years old, from about 50 to about 55 years old, from about 55 to about60 years old, from about 60 to about 65 years old, from about 65 toabout 70 years old, from about 70 to about 75 years old, from about 75to about 80 years old, from about 80 to about 85 years old, from about85 to about 90 years old, from about 90 to about 95 years old or fromabout 95 to about 100 years old. In one embodiment, the human is achild. In one embodiment, the human is a female.

In other embodiments, the subject is a non-human animal, and thereforethe invention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal.

Kits

The invention provides kits that can simplify the administration of anyagent described herein. An exemplary kit of the invention comprises anycomposition described herein in unit dosage form. In one embodiment, theunit dosage form is a container, such as a pre-filled syringe, which canbe sterile, containing any agent described herein and a pharmaceuticallyacceptable carrier, diluent, excipient, or vehicle. The kit can furthercomprise a label or printed instructions instructing the use of anyagent described herein. The kit may also include a lid speculum, topicalanesthetic, and a cleaning agent for the administration location. Thekit can also further comprise one or more additional therapeutic agentsdescribed herein. In one embodiment, the kit comprises a containercontaining an effective amount of a composition of the invention and aneffective amount of another composition, such those described herein.

In some embodiments, the additional therapeutic agent is an adjunctivetherapy that is used in, for example, the treatment of CDI as describedherein. In some embodiments, the additional therapeutic agent ismetronidazole (e.g. FLAGYL), fidaxomicin (e.g. DIFICID), or vancomycin(e.g. Vancocin), rifaximin, fecal bacteriotherapy, charcoal-basedbinders/adsorbents (e.g. DAV132), probiotic therapy (see, e.g., Intnat'lJ Inf Dis, 16 (11): e786, the contents of which are hereby incorporatedby reference, illustrative probiotics include Saccharomyces boulardii;Lactobacillus rhamnosus GG; Lactobacillus plantarum 299v; Clostridiumbutyricum M588; Clostridium difficile VP20621 (non-toxigenic C.difficile strain); combination of Lactobacillus casei, Lactobacillusacidophilus (Bio-K+CL1285); combination of Lactobacillus casei,Lactobacillus bulgaricus, Streptococcus thermophilus (Actimel);combination of Lactobacillus acidophilus, Bifidobacterium bifidum(Florajen3); combination of Lactobacillus acidophilus, Lactobacillusbulgaricus delbrueckii subsp. bulgaricus, Lactobacillus bulgaricuscasei, Lactobacillus bulgaricus plantarum, Bifidobacterium longum,Bifidobacterium infantis, Bifidobacterium breve, Streptococcussalivarius subsp.thermophilus (VSL#3)) and antibody or other biologictherapy (e.g. monoclonal antibodies against C. difficile toxins A and Bas described in N Engl J Med. 2010; 362(3):197, the content of which arehereby incorporated by reference in their entirety; neutralizing bindingproteins, for example, arranged as multimers, which are directed to oneor more of SEQ ID NOs. recited in United States Patent Publication No.2013/0058962 (e.g. one or more of SEQ ID Nos.: 59, 60, 95, 67, 68, and87), the contents of which are hereby incorporated by reference); orany-neutralizing binding protein directed against C. difficile binarytoxin. In some embodiments, any of the penicillins and cephalosporinsdescribed herein may be the additional therapeutic agent.

EXAMPLES Example 1 SYN-004 Microbiome Protection from Oral AmoxicillinMicrobiome Damage

SYN-004 was formulated as an enteric-coated pellet that releases at pHsof 5.5 and higher. Therefore, SYN-004 is protected from low pH, similarto what is found in the stomach, and released at pHs greater than 5.5,similar to the pH in the duodenum (pH 5.9-6.6). Release of SYN-004 isexpected to continue throughout the small intestine, i.e., the jejunum(pH 6.6-7.4), the ileum (pH 7.3-7.8), and/or cecum (pH 5.6-5.9).

The formulation used for this study is as follows:

TABLE 1 Composition of P3A Delayed-Release Capsules, 75 mg and 25 mg,and Placebo Capsule 75 mg 25 mg Placebo Capsule Capsule Capsule % % %Component mg Total mg Total mg Total Sucrose sphere 110.8 23.3 36.9 23.3139.8 29.5 Hydroxypropylcellulose 166.3 35.0 55.4 35.0 209.6 44.2EUDRAGIT ® 98.9 20.8 33.0 20.8 98.7 20.8 L 30 D-55 P3A 75.0 15.8 25.015.8 — — Buffer salts 7.5 1.6 2.5 1.6 9.4 2.0 Glyceryl monostearate 4.91.0 1.6 1.0 4.9 1.0 Polysorbate-80 2.0 0.4 0.7 0.4 2.0 0.4 Triethylcitrate 9.9 2.1 3.3 2.1 9.9 2.1 Subtotal 475.3 100.0 158.4 100.0 474.3100.0 Hard gelatin capsule 96.0 96.0 96.0 #0 or Hydroxypropylmethylcellulose (HPMC) capsule Total 571.3 254.4 570.3and as described in PCT/US15/54606, the entire contents of which areincorporated by reference.

In vitro dissolution studies revealed that the current formulation ofSYN-004 is released in a pH-dependent manner and requires 1-3 hours forcomplete release, while transit time through the small intestine isapproximately 3 hrs+1 hr SEM after entering the duodenum. See, e.g. U.S.patent application Ser. No. 14/878,155, the entire contents of which arehereby incorporated by reference. These data suggest that SYN-004 willbe released in a sustained manner throughout the proximal and distalsmall intestine. Orally-delivered antibiotics such as amoxicillin areabsorbed in the proximal small intestine such as the duodenum and thejejunum, but not in the ileum (Barr, et al., 1994).

A study was performed using normal piglets to determine if SYN-004, whendelivered orally with oral amoxicillin, functions to protect themicrobiome from amoxicillin-induced dysbiosis. The study also tested ifSYN-004 affected the absorption of amoxicillin from the GI tract ofanimals (TABLE 2).

TABLE 2 Piglet study design Group Antibiotic (N = 5) Antibiotic DeliverySYN-004 1 Amoxicillin Oral, BID, each dose None Pig 1, 2, 3, suspension20 mg/kg 4, 5 (40 mg/kg/day) 7 am, 5 pm 2 Amoxicillin Oral, BID, eachdose 1 size 0 capsule Pig 6, 7, 8, suspension 20 mg/kg (75 mg), QID 9,10 (40 mg/kg/day) 7 am, 5 pm 7am, 12 pm, 5 pm, 10 pm

A total of ten, two-month old Yorkshire piglets, approximately 20 kgeach, were used for this study. All 10 animals were treated with oralamoxicillin twice a day for a total of 7 days, and one cohort of 5animals was also treated with oral SYN-004 four times a day for a totalof 9 days. The SYN-004 treatment was started the day before amoxicillintreatment and continued for a day after amoxicillin was stopped (FIG.1).

Two pre-treatment fecal samples were obtained, the first 4 days afterthe animals arrived at the animal treatment facility (Day −7), and thesecond 7 days after arrival (Day −4). An additional 3 fecal samples werecollected at Day 4, Day 8, and Day 9. The fecal samples were collectedusing the OMNIgene GUT sample collection kits (OMR-200, DNA Genotek,Ontario, Canada) and stored at room temperature away from light untilall samples were collected. DNA isolated from the fecal samples wassubjected to deep sequencing of the intestinal microbiome and analyses.

On Study Day 0, Group 2 (Pigs 6-10) received one size 0 capsule ofSYN-004; containing 75 mg of SYN-004, orally, four times a day at 7 am,12 pm, 5 pm, and 10 pm for a total of 9 days. Pigs were fed 3 times aday, after SYN-004 dosing at 7 am, after SYN-004 dosing at 12 pm, andafter SYN-004 dosing at 5 pm. Beginning on Study Day 1, Groups 1 and 2(Pigs 1-10) received oral amoxicillin (fruit flavored oral suspension,Sandoz, NDC: 0781-6157-46, Lot #EY9130; 20 mg/kg) twice a day at 7 amand at 5 pm, for a total of 7 days. Animals received the amoxicillinfirst, followed by the SYN-004, then feeding.

On Day 2, after 4 amoxicillin doses, animals were bled and serumcollected. Blood was collected aseptically from the vena cava fromanesthetized animals. Three blood draws were performed, at 1 hr, 3 hrs,and 8 hrs after amoxicillin administration. A Telazol cocktail wasadministered intramuscularly at a minimal dose (1 mL or less per 50 lbs)to achieve light anesthesia/sedations. At each timepoint, approximately9 mL of blood was collected into a serum separator vacutainer tube.After coagulation, samples were centrifuged and the serum wastransferred to a cryovial and stored at −80° C. until shipment to theevaluation laboratory (Center for Anti-Infective Research andDevelopment, Hartford Hospital, Hartford, Conn.).

FIG. 1 shows a timeline of piglet dosing. Animals received SYN-004 for 9days starting on Day 0. Animals received oral amoxicillin for 7 daysstarting on Day 1. Stool was collected at 5 times, Day −7, Day −4, Day4, Day 8, and Day 9. Blood was collected at 3 times during Day 2.

Amoxicillin levels in the pig serum were quantified using a modificationof a validated HPLC-based assay (Du et al., 2005). A standard curve wasprepared in negative control pig serum and had 6 points ranging from 1to 30 ug/mL of amoxicillin. The assay was linear over a range of 1 to 30ug/mL (R=0.999). Interday coefficients of variation for the low (1.5ug/mL) and high (20 ug/mL) quality control samples were 3.9% and 4.9%respectively. Interday coefficients of variation were 3.8% and 2.8%,respectively. Peak height was used to integrate all the peaks. SigmaPlot was used to calculate drug concentrations and a −1 weighting factorwas used. An interfering peak was overcome by raising the standard curvefrom 0.25 to 1 ug/mL for the amoxicillin. The limit of detection of theassay was 1.5 ug/mL. The amoxicillin levels were reported as the meanand standard deviation (FIG. 2). The amoxicillin levels at one hour were8.9±0.8 for amoxicillin alone and 8.4±1.2 for amoxicillin+SYN-004. At 3hours, the amoxicillin levels were 3.9±0.3 for the amoxicillin alone and2.3±0.6 for the amoxicillin+SYN-004. At 8 hours, the amoxicillin levelswere 1.5±0.3 for the amoxicillin alone, and 1.8±0.3 for theamoxicillin+SYN-004. These data demonstrate that oral SYN-004 did notprevent the absorption of amoxicillin from the GI tract. Therefore,these data verify that SYN-004 did not degrade the amoxicillin in the GItract prior to amoxicillin absorption, suggesting that the amoxicillinwas absorbed before SYN-004 was released into the GI tract.

DNA was isolated from the fecal samples and subjected to whole genomeshotgun sequencing using an Illumina HiSeq system with a target of 20million 100 bp single reads per sample.

Sequenced datasets were taxonomically classified using the GENIUS®software package (Hasan et al., 2014, Lax et al., 2014).

The median similarity based on the relative bacterial strain abundancewas calculated. The percent similarity from Day −7 to Day 9 of theamoxicillin and amoxicillin+SYN-004 groups was compared (FIGS. 3 and 4).The diversity of the microbiome decreased from Day −7 to Day −4 in bothgroups. As the animals were in the process of acclimating and were notyet treated at Day −4, these data suggest that the microbiome waschanging based on the new environment. Animals began amoxicillintreatment on Day 1 and by Day 4, the microbiome in theAmoxicillin+SYN-004 group had stabilized to a median similaritycomparable to that of Day −4. Notably, the microbiome of theAmoxicillin-alone treated pigs continued to lose diversity throughoutthe duration of the study. These data demonstrate that SYN-004 preventedthe loss of diversity in the microbiome caused by amoxicillin treatment.

FIG. 3 shows strain relative abundance percent similarity. The medianpercent similarity based on the relative abundance of the bacterialstrains identified from sequence analysis of the fecal DNA samples wascompared for the amoxicillin alone group (n=5) and theamoxicillin+SYN-004 group (n=5) from Day −7 to Day 9. Amoxicillin aloneis displayed as the solid line, and Amoxicillin+SYN-004 is displayed asthe dashed line.

FIG. 4 shows strain relative abundance percent similarity boxplot. Themedian percent similarity based on the relative abundance of thebacterial strains identified from sequence analysis of the fecal DNAsamples was compared for the amoxicillin alone group (n=5) and theamoxicillin+SYN-004 group (n=5) from Day −7 to Day 9. Amoxicillin aloneis displayed as the gray box, and Amoxicillin+SYN-004 is displayed asthe white box. The boxplot displays the median (line), the quartiles(box) and the range (vertical lines).

Heatmaps of the bacterial taxa were constructed based on the relativeabundance of each bacterial strain and organized chronologically bystudy day and by treatment group (FIG. 5).

The abundance of some bacterial species decreased in the amoxicillinalone groups by Day 4 while these groups were maintained in theamoxicillin+SYN-004 group. Similarly, some bacterial species increasedin abundance in the amoxicillin alone group while the same bacteria didnot overgrow when SYN-004 was present with amoxicillin. These datademonstrate that SYN-004 protected the microbiome from the effects ofamoxicillin.

FIG. 5 shows strain abundance heat map. Heatmaps of the bacterial taxawere constructed based on the relative abundance of each bacterialstrain and organized chronologically by study day and by treatmentgroup. The groups are labeled on the left side of the figure,Amoxicillin (Amox) and Amoxicillin+SYN-004 (Amox+SYN-004), and thetimepoints are indicated by the different colored bars on the left sideof the figure according to the Collection Day key. The individualbacterial strains are displayed on the bottom, and the bacterial growthclass of each strain is indicated on the top according to the GrowthClass key. The individual animals are indicted on the right side of thefigure. The white boxes on the right side of the figure indicatebacterial strains that were decreased in the Amoxicillin alone group butretained in the Amoxicillin+SYN-004 group. The green boxes on the leftside of the figure indicate bacterial strains that became more abundantin the Amoxicillin alone group but that did not overgrow in theAmoxicillin+SYN-004 group.

A statistical analysis was performed to determine the probability thatthe microbiomes before and after antibiotic treatment remained the sameor were different. The microbiome sequence data were analyzed using aparameterization of the Dirichlet-Multinomial distribution (La Rosa etal., 2012) to perform a Likelihood Ratio Test. The pretreatment Day −4and the post-treatment Day −9 microbiomes of the Amoxicillin alone andAmoxicillin+SYN-004 treatment groups were compared. The p value obtainedcomparing the Amoxicillin alone group before and after amoxicillintreatment was p=0.0000000010521, indicating that the two groups weresignificantly different. In contrast, the p value obtained comparing theAmoxicillin+SYN-004 group was 0.9970586680662, indicating that these twogroups were not significantly different. These data demonstrate thatSYN-004 protected the microbiome from amoxicillin-mediated damage.

Example 2 SYN-004 Multi-Particulates, Additional SYN-004 Formulationsand Their In Vitro Characterization

Three additional modified-release formulations of SYN-004 were generatedand tested. The starting material for the formulations wasSYN-004-coated sucrose pellets that lacked the outer, enteric-coating.P3A layered pellets were produced by spray application of P3A drugsubstance using hydroxypropylcellulose (HPC) or hydroxypropylmethylcellulose (HPMC) as a binder excipient, water as a solvent, and sucrosespheres as starting material. The spray application was performed usinga fluid bed system over six work shifts, in order to achieve a finalactive pharmaceutical agent (API) percentage of at least 15%. After thesixth work shift of spray application of the P3A/HPC mixture, the P3Alayered pellets were dried overnight at room temperature on trays, thensifted through a 1.4 mm sieve prior to bulk packaging in polyethylene(PE) bags and PE containers. The drug-layered pellets were stored at5±3° C. for further processing. For example, in some embodiments, theP3A layered pellets were coated with different coatings to achievespecific enzyme release profiles.

The three new formulations utilized different coatings to obtainmodified enzyme release profiles (FIG. 6). The different coatingsincluded an enteric coating that released at pHs of >6.2 or an entericcoating that released at pHs of >6.7. The third type of coating was anosmotic-rupture coating that released the enzyme within a specifiedtimeframe, between 2-4 hours after ingestion. The three formulationswere characterized in vitro for physical appearance, composition, andenzyme dissolution profiles. The three formulations with the mostpromising profiles were selected for evaluation with oral amoxicillin ina pig model.

Enteric-Coating SYN-004 Formulation with Release at pHs >6.2

The SYN-004-coated sucrose pellet starting material was coated with amixture of Eudragit L100, Eudragit S100, and triethyl citrate at a ratioof 72.7/18.2/9.1. The parameters of the spray coating (FIG. 6) was usinga Niro-Aeromatic Lab Fluid Bed Dryer, Model MP-1 with a bowl size of 3.5inches, air distribution with a Mod B Plate, a Schlick 970 (tall) nozzlewith a 1.2 mm liquid tip size, and a column gap of 10 mm. The EudragitL100, Eudragit S100, and triethyl citrate at a ratio of 72.7/18.2/9.1was dissolved in isopropanol and water at a 95/5 ratio. First theisopropanol and water were mixed and then the Eudragits were added,after which the triethyl citrate was added. The mixture was stirreduntil dissolved for at least 30 minutes following the addition of thetriethyl citrate. The operating parameters of the fluid bed dryer were agas flow rate of 35 CFM, inlet temperature of 28° C., inlet dew point of8.6° C., atomizer pressure of 2.7 bar, a spray rate of 2.3 g/min, and abed temperature of 25° C. The fluid bed process performance was a totalsolution sprayed of 612 g, a run time of 4 hrs and 25 minutes, a beddump of 105.1 g, a final coat weight estimate of 35% and a coatingefficiency of 97.2%. Samples were dried at 400° C. for 2 hours. Sampleswere collected at intermediate coating weights of 25% and 30% forcharacterization along with the coat weight of 35%.

The coated particles were characterized based on coat thickness vs coatweight and mass fraction (weight %) vs particle size (FIG. 7, panels Aand B). The collected particles were confirmed to have estimated coatweights of 25% with an average coat thickness of approximately 70 um,30% with an average 80 um coat thickness, and 35% with an average 100 umcoat thickness. The final product particles (35% coating efficiency)were characterized based on the mass fraction vs particle size and itwas found that the midpoint of the particle sizes (D₅₀) was 1390 um. Thecoated particles were also characterized by scanning electron microscopy(FIGS. 8 and 9). The particles appeared smooth and uniformly coated withsized of approximately 1.4 mm. Cross sections of the particles (n=6 foreach coating %) allowed calculation of the coating thicknesses. Thecalculated coating thicknesses were 69.7 um for the 25%, 77.9 um for the30% and 99.7 um for the 35%, similar to what was observed in the coatthickness vs coat weight evaluation (FIG. 7). Additional scanningelectron microscopy characterization of the 35% particles displayed auniform surface and a 50× magnified view of a cross sectioned particleclearly displayed the sucrose core, the SYN-004 layer over the core, andthe outer Eudragit coating (FIG. 9). Based on these data, the 35%pH >6.2 particles were chosen as the prototype for further testing.

Enteric-Coating SYN-004 Formulation with Release at pHs >6.7

The SYN-004-coated sucrose pellet starting material was coated with amixture of Eudragit L100, Eudragit S100, and triethyl citrate at a ratioof 30/60.9/9.1. The parameters of the spray coating (FIG. 6) was using aNiro-Aeromatic Lab Fluid Bed Dryer, Model MP-1 with a bowl size of 3.5inches, air distribution with a Mod B Plate, a Schlick 970 (tall) nozzlewith a 1.2 mm liquid tip size, and a column gap of 10 mm. The EudragitL100, Eudragit S100, and triethyl citrate at a ratio of 72.7/18.2/9.1was dissolved in isopropanol and water at a 95/5 ratio. First theisopropanol and water were mixed and then the Eudragits were added,after which the triethyl citrate was added. The mixture was stirreduntil dissolved for at least 30 minutes following the addition of thetriethyl citrate. The operating parameters of the fluid bed dryer were agas flow rate of 35 CFM, inlet temperature of 29° C., inlet dew point of7.5° C., atomizer pressure of 2.5 bar, a spray rate of 2.4 g/min, and abed temperature of 25° C. The fluid bed process performance was a totalsolution sprayed of 628.7 g, a run time of 4 hrs and 24 minutes, a beddump of 105.1 g, a final coat weight estimate of 35% and a coatingefficiency of 99.6%. Samples were dried at 35° C. for 2 hours. Sampleswere collected at intermediate coating weights of 25% and 30% forcharacterization along with the 35%.

The coated particles were characterized based on coat thickness vs coatweight and mass fraction (weight %) vs particle size (FIG. 10, panels Aand B). Coating was more efficient than expected and the estimated coatweights were 30% (referred to as the expected 25%), 35% (referred to asthe expected 30%), and 40% (referred to as the expected 35%), with coatthicknesses of approximately 65 um, 85 um, and 110 um, respectively. Thefinal product particles (40% coating efficiency) were characterizedbased on the mass fraction vs particle size and it was found that themidpoint of the particle sizes (D₅₀) was 1388 um. The coated particleswere also characterized by scanning electron microscopy (FIGS. 11 and12). The particles appeared smooth and uniformly coated with sizes ofapproximately 1.5 mm. Cross sections of the particles (n=6 or n=10 foreach coating %) allowed calculation of the coating thicknesses. Thecalculated coating thicknesses were 68.5 um for the 25%, 85.2 um for the30% and 113 um for the 35%, similar to what was observed in the coatthickness vs coat weight evaluation (FIG. 10). Additional scanningelectron microscopy characterization of the 35% particles displayed auniform surface and a 50× magnified view of a cross sectioned particleclearly displayed the sucrose core, the SYN-004 layer over the core, andthe outer Eudragit coating (FIG. 12).

Based on these data, the 35% pH >6.7 particles were chosen as theprototype for further testing.

Osmotic-Rupture Coating SYN-004 Formulation with Timed Release

The SYN-004-coated sucrose pellet starting material was coated with amixture of 71.4% pulverized croscarmellos sodium (AcDiSol, FMCBiopolymer), 28.6% hydroxyproplycellulose (HPC) in 100 proof ethylalcohol. This layer was referred to as the sweller layer. The HPC wasadded to ⅔ of the ethyl alcohol and the AcDiSol was added to ⅓ of theethyl alcohol. The solution was high shear mixed for 3 minutes at 4000rpm. The parameters of the spray coating (FIG. 6) was using aNiro-Aeromatic Lab Fluid Bed Dryer, Model MP-1 with a bowl size of 3.5inches, air distribution with a Mod B Plate, a Schlick 970 (tall) nozzlewith a 1.2 mm liquid tip size, and a column gap of 10 mm. The operatingparameters of the fluid bed dryer were a gas flow rate of 35 CFM, inlettemperature of 32° C., inlet dew point of 6.1° C.,-atomizer pressure of2.5 bar, a spray rate of 3.4 g/min, and a bed temperature of 28° C. Thefluid-bed process performance was a total solution sprayed of 385 g, arun time of 1 hr and 53 minutes, a bed dump of 119.1 g, a final coatweight estimate of 37% and a coating efficiency of 82%. No intermediatesamples were collected.

The next step was to add the osmotic rupture coating to the SYN-004particles coated with the sweller layer. The osmotic rupture coatingcomposition was 75% Aquacoat ECD (ethylcellulose dispersion, FMCBiopolymer), and 25% triethyl citrate (TEC) in water. The TEC was addedto the Aquacoat ECD and the residual TEC was washed with water and addedto solution. The suspension was stirred throughout the run. Theparameters of the spray coating (FIG. 6) was using a Niro-Aeromatic LabFluid Bed Dryer, Model MP-1 with a bowl size of 3.5 inches, airdistribution with a Mod B Plate, a Schlick 970 (tall) nozzle with a 1.2mm liquid tip size, and a column gap of 10 mm. The operating parametersof the fluid bed dryer were a gas flow rate of 35 CFM, inlet temperatureof 51° C., inlet dew point of 8.3° C., atomizer pressure of 2.5 bar, aspray rate of 2.0 g/min, and a bed temperature of 35° C. The fluid bedprocess performance was a total solution sprayed of 146 g, a run time of1 hr and 11 minutes, a bed dump of 124, a final coat weight estimate of13.5% and a coating efficiency of 66%. Intermediate samples werecollected at 7.3%, 9.1%, 10%, and 11.5%.

Following coating, the particles were cured. Multiple curingtemperatures and times were evaluated for osmotic rupture (burst time)and beta-lactamase enzyme activity. The initial analyses comparedparticles with 10% or 11.5% osmotic coat weights with cure temperaturesof 50° C. or 60° C., and cure times of 2, 5, and 8 hours (FIG. 13).Pellets were added to a 50 mM KH₂PO₄ pH 6.2 buffer at room temperaturewithout stirring, images of the pellets were taken every 5 minutes toevaluate particle disruption as visual disruption of the coating,significant deformation of the particles and the presence of externalAcDiSol. The data demonstrated that the 10% and 11.5% coat weights werenot of sufficient thickness to reach the desired 4 hours particle burstdelay, as the particles started to appear broken between 2 and 3 hours.In addition, the cure time and temperature of 60° C. for 2 hoursappeared optimal. Based on these data, the 10% SYN-004 pellets wererecoated to 11.4% and 13.5% with a cure temperature of 60° C. for 2hours and retested as described (FIG. 14). Pellets with the 13.5%coating remained intact for over 5 hours with 50% rupture occurring at 8hours, while the 10% and 11.4% began to break at 2.5 or 3 hours with 50%rupture occurring at 4 hours. The appearance of the 10% and 13.5%particles after soaking for 0 to 8.5 hours is displayed in FIG. 15.

The coated particles were characterized based on coat thickness vs coatweight and mass fraction (weight %) vs particle size (FIG. 16). Coatingwas as expected with coat weights ranging from 7% to 13%, andthicknesses of 20 um to 50 um. The final product particles (13.5%coating weight) were characterized based on the mass fraction vsparticle size and it was found that the midpoint of the particle sizes(D₅₀) was 1432 um. The coated particles were also characterized byscanning electron microscopy (FIGS. 17 and 18). The particles appearedsmooth and uniformly coated with sizes of approximately 1.5 mm. Crosssections of the particles (n=10 for each coating %) allowed calculationof the coating thicknesses. The calculated coating thicknesses were 30.8um for the 10% coat weight, 41.3 um for the 11.5% coat weight, and 48 umfor the 13.5% coat weight. Additional scanning electron microscopycharacterization of the 13.5% coat weight particles displayed a uniformsurface and a 50× magnified view of a cross sectioned particle displayedthe sucrose core, the SYN-004, and coating layers (FIG. 18).

To verify that the conditions chosen to cure the osmotic coating did notaffect SYN-004 biological activity, an additional study was performed.Osmotic rupture, 13.5% coat weight SYN-004 pellets, under differentcuring conditions were evaluated for retention of biological activity(FIG. 19). Osmotic rupture pellets were added to a pH 6.8 potassiumphosphate buffer and stirred overnight to ensure removal of the entirecoating. Aliquots of the buffer were analyzed for SYN-004 biologicalactivity using the CENTA chromogenic microtiter plate assay. The datademonstrate that cure times ranging from 8 hours at 50° C. or 2 hours at60° C. did not affect SYN-004 biological activity. The SYN-004 startingmaterial, the uncoated pellets, displayed 84.0%±15.2% activity, theuncured particles, 94.5+12.2% activity, 50° C. for 2 hrs, 84.0+6.2%activity, 50° C. for 5 hr, 75.8+6.4%, 50° C. for 8 hr, 83.9+1.9%activity, and 60° C. for 2 hrs, 86.1+1.4% activity.

Based on these data, the 13.5%, cured at 60° C. for 2 hours, osmoticrupture particles were chosen as the prototype for further testing.

The criteria for identifying a promising modified-release formulation ofSYN-004 for oral use with oral antibiotics was to identify SYN-004formulation or formulations that have the desired enzyme release profileto maximize antibiotic bioavailability while minimizing the antibiotic'seffect on the intestinal microflora (FIG. 20). The three SYN-004formulations generated as described in Example 2 were furthercharacterized. The three formulations chosen were: 1) enteric-coated pH6.2 release, 2) enteric-coated pH 6.7 release, and 3) an osmotic ruptureformulation. The characteristics of these formulations are displayed inTABLE 3.

TABLE 3 Biological Release characteristics Activity Formulation ReleaseCoating Lag Duration Total Compared Prototypes pH %/thickness 2 hr 4 hrTime (hr) (hr) to SYN-004 SYN-004 5.5 ~55 um 0-1% 20-50% NA 1.3 2.25100% (Control) Enteric 6.2 6.2 35%/100 um  0-10%  5-15% NA 2.0 4 + 2135% Enteric 6.7 6.7 35%/113 um 0-1% 0-5% NA 8-10 4 + 8-10 120% OsmoticNA 13.5%/48 um 0-5% 5.50% 3 hr 3   6   105%

The three formulations were evaluated in vitro to characterize theirdissolution profiles and to verify that the SYN-004 enzyme retainedbiological activity in each formulation (FIGS. 21 and 22). For eachformulation, a total of 7.5 mg of SYN-004 active agent was incubated in25 ml of a pH 2.0 solution (0.01N HCl) for 2 hours to mimic theconditions of the stomach following ingestion. The pH was then increasedto 5.5 (total volume 75 ml in potassium phosphate) for an additional 2hours. The pH was then adjusted to 6.5 using 10N KOH. All incubationswere performed at 37.5° C. with agitation of 250 rpm. For each sample,20 ul was collected into a 2 ml volumetric post auto sampler at theindicated time points. Samples were evaluated for protein concentrationusing absorbance at 280 nm and for SYN-004 biological activity using theCENTA chromogenic assay. The Enteric pH 6.2 formulation showed minimalrelease in acidic environments with a duration of release ofapproximately 2 hours. The Enteric pH 6.7 formulation showed minimalrelease in acidic environments followed by a duration of release ofapproximately 8-10 hours. The Osmotic Rupture formulation providingpH-independent release, displayed a 3 hour lag followed by a duration ofrelease of 3 hours. The original SYN-004 formulation (Enteric, pH 5.5)showed minimal release in acidic environments followed by a duration ofrelease of 1.3 hours. The data demonstrate that all formulationsmaintain SYN-004 biological activity for the duration of the three-stagedissolution test for 24 hours. The formulations have the desired releasecharacteristics for testing in an animal model with an oral antibioticssuch as amoxicillin.

Example 3 Preparation of Capsules filled with SYN-004 Formulations forIn Vivo Evaluations

The three SYN-004 formulation pellets, as described in Example 2, andthe original SYN-004 formulation pellets (Enteric 5.5) as described inExample 1, were used to fill size 0 gelatin capsules to achieve a doseof 50 mg of SYN-004 active agent (TABLE 4). The list of components andthe amounts in these capsules are provided in TABLE 5 below. Avolumetric fill was used with a 100% weight sort and a +3% targetrejection limit. There was less than a 3% RDS fill variation. Theaverage mass of the empty gelatin capsules was 93 mg.

TABLE 4 Formulation Target Fill Average Prototypes Capsule Weight FillWeight % Capsules Rejected (50 mg active) Color (mg) (mg) % Target % RSD(±3% Limits) SYN-004 clear 298 297 99.9% 1.0% 0.0% (Control) Enteric 6.2green 360 359 99.9% 1.4% 0.9% Enteric 6.7 orange 401 402 100.2%  1.2%2.3% Osmotic white 447 447  100% 1.1% 3.2%

TABLE 5 Composition of osmotic rupture, enteric pH 6.2, and enteric pH6.7 SYN-004 50 mg active capsules Osmotic Enteric Enteric Rupture pH 6.2pH 6.7 % % % Component mg Total mg Total mg Total Sucrose sphere 73.716.5 73.7 20.5 73.7 18.4 Hydroxypropylcellulose 140.0 31.3 110.5 30.7110.5 27.5 SYN-004 50.0 11.2 50.0 13.9 50.0 12.5 EUDRAGIT ® L100 — —87.5 24.3 49.1 12.2 EUDRAGIT ® S100 — — 21.9 6.1 98.2 24.5 AcDiSol 110.024.8 — — — — Aquacoat ECD 29.3 6.6 — — — — Buffer salts 5.0 1.1 5.0 1.45.0 1.3 Talc 38.2 8.6 — — — — Triethyl citrate — — 10.9 3.0 14.7 3.7Subtotal 446 100.0 360 100.0 401 100.0 Hard gelatin capsule #0 93.0 93.093.0 Total 539 445 494

The dissolution of the SYN-004 pellets from the capsules was evaluatedand compared to the data obtained from dissolution of the SYN-004pellets prior to encapsulation (FIG. 23). The dissolution study wasperformed as described previously, however, instead of using 7.5 mg ofpellets, one capsule containing 50 mg of active SYN-004 was used.Briefly, one capsule of each formulation, except SYN-004 original(Enteric pH 5.5) was incubated in a pH 2.0 solution (0.01N HCl) for 2hours to mimic the conditions of the stomach following ingestion. The pHwas then increased to 5.5 for an additional 2 hours. The pH was thenadjusted to 6.5 using 10N KOH. All incubations were performed at 37.50°C. with agitation of 250 rpm. For each sample, 20 ul was collected intoa 2 ml volumetric post auto sampler at the indicated time points.Samples were evaluated for protein concentration using absorbance at 280nm. The data demonstrate that no damage to the pellets occurred duringcapsule filling. A slower than expected release rate was observed forthe Enteric pH 6.2 and the Enteric pH 6.7 formulations.

Following encapsulation, the formulations retained the desired releasecharacteristics for testing in an animal model with an oral antibioticssuch as amoxicillin.

Example 4 Evaluation of the Modified-Release Formulations of SYN-004 inNormal Piglets

A study was performed using normal piglets to compare the three,modified-release formulations of SYN-004 as described in Example 2, andthe original SYN-004 formulation (Enteric pH 5.5) as described inExample 1, delivered orally with oral amoxicillin, to evaluate theeffect on amoxicillin serum levels and protection of the microbiome fromamoxicillin-induced dysbiosis (FIG. 24).

A total of 25, two month old Yorkshire piglets, approximately 20 kgeach, were used for this study. All 25 animals were treated with oralamoxicillin twice a day for a total of 7 days, in addition, Groups 2-5received SYN-004 four times a data starting the day before amoxicillintreatment for a total of 9 days (TABLE 6).

TABLE 6 Piglet Study Design Group SYN-004 Antibiotic (N = 5) SYN-004Delivery Delivery 1 None None 2 SYN-004 1 capsule Amoxicillin (originalformula) (50 mg active) suspension Clear capsules QID (40 mg/kg/day) 3SYN-004 Formulation #2 7 am, 12 pm, 5 pm Oral, each dose Green Capsulesand 10 pm 20 mg/kg BID 4 SYN-004 Formulation #3 7 am, 5 pm OrangeCapsules 5 SYN-004 Formulation #4 White Capsules

Three pre-treatment fecal samples were obtained, at Day −4, Day −2, andDay 0 (prior to SYN-004 treatment. The fecal samples were collectedusing the OMNIgene GUT sample collection kits (OMR-200, DNA Genotek,Ontario, Canada) and stored at room temperature away from light untilall samples were collected. DNA isolated from the fecal samples wassubjected to deep sequencing of the intestinal microbiome and analyses.Additional fecal samples were collected at Day −2, and Day 4. Thesesamples were collected into 50 ml conical tubes and quickly frozen andstored at −80° C. These samples were submitted for amoxicillinquantification.

On Study Day 0, Groups 2-5 (Pigs 6-25) received one size 0 capsule ofone of each of the four SYN-004 formulations, containing 50 mg ofSYN-004, orally, four times a day at 7 am, 12 pm, 5 pm, and 10 pm for atotal of 9 days. Pigs were fed 3 times a day, after SYN-004 dosing at 7am, after SYN-004 dosing at 12 pm, and after SYN-004 dosing at 5 pm.Beginning on Study Day 1, all groups Groups 1-5 (Pigs 1-25) receivedoral amoxicillin (fruit flavored oral suspension, Sandoz, NDC:0781-6157-46, Lot #FB0703; 20 mg/kg) twice a day at 7 am and at 5 pm,for a total of 7 days. Animals received the amoxicillin first, followedby the SYN-004, then feeding.

On Day 3, after 5 amoxicillin doses, animals were bled and serumcollected. Blood was collected aseptically from the vena cava fromanesthetized animals. Three blood draws were performed, at 1 hr, 3 hrs,and 6 hrs after amoxicillin administration. A Telazol cocktail wasadministered intramuscularly at a minimal dose (1 mL or less per 50 lbs)to achieve light anesthesia/sedations. At each timepoint, approximately9 mL of blood was collected into a serum separator vacutainer tube.After coagulation, samples were centrifuged and the serum wastransferred to a cryovial and stored at −80° C. until shipment to theevaluation laboratory (Center for Anti-Infective Research andDevelopment, Hartford Hospital, Hartford, Conn.).

Amoxicillin levels in the pig serum are quantified using a modificationof a validated HPLC-based assay (Du et al., 2005). A standard curve isprepared in negative control pig serum and had 6 points ranging from 1to 30 ug/mL of amoxicillin. Sigma Plot is used to calculate drugconcentrations and a −1 weighting factor is used. The limit of detectionof the assay is 1.5 ug/mL.

DNA is isolated from the fecal samples and subjected to whole genomeshotgun sequencing using an Illumina HiSeq system with a target of 20million 100 bp single reads per sample. DNA isolation and sequencing areperformed by Hudson Alpha Genomic Services Laboratory (Huntsville,Ala.). Sequenced datasets are taxonomically classified using the GENIUS®software package (Hasan et al., 2014, Lax et al., 2014) by CosmosID,Inc. (Rockville, Md.).

The resulting data are expected to demonstrate that each formulation ofSYN-004 does not prevent the absorption of amoxicillin from the GItract, suggesting that the amoxicillin was absorbed before SYN-004 wasreleased into the GI tract. In addition, the resulting data are expectedto demonstrate that each formulation of SYN-004 protected the microbiomefrom the damage caused by amoxicillin treatment.

Example 5 Formulations

The beta-lactamase is formulated for release in a location in the GItract in which it deactivates residual oral antibiotic residue,specifically for release in a location in the GI tract that is distal tothe release of the orally administered antibiotic.

P3A is formulated by combining P3A with a latex, or other polymer, and aparticulate, micro-encapsulated enzyme preparation is formed. Themicrospheres may then be covered with a pH-dependent enteric coating. Nosucrose core is required and this allows for higher drug loading perpellet and therefore a smaller capsule size for therapy. Formulationsare developed to produce particles that have enteric functionality (notreleased in the stomach, complete release in the distal small intestine)built into the matrix itself, to reduce excipient load. If theformulation shows good retention of activity and stability, butinsufficient protection from acidic conditions, enteric coating isapplied to the particulates.

A variety of approaches for generating particulates (such asmicrospheres, aggregates, other) that are amenable to the inclusion ofproteins may be used. These approaches involve at least two phases, onecontaining the protein, and one containing a polymer that forms thebackbone of the particulate. For example, coacervation, where thepolymer is made to separate from its solvent phase by addition of athird component, or multiple phase emulsions, such as water in oil inwater (w/o/w) emulsion where the inner water phase contains the protein,the intermediate organic phase contains the polymer, and the externalwater phase stabilizers that support the w/o/w double emulsion until thesolvents can be removed to form the microspheres may be used.

In another approach, the protein and stabilizing excipients (e.g.,hydroxyproplyl methylcellulose acetate succinate (HPMCAS) type MF;Aquacoat (FMC), sodium stearyl fumarate; trehalose, mannitol, Tween 80,polyvinyl alcohol, and/or others) are combined and then the mixture fromaqueous solution is sprayed, particles form and are collected. Theparticles are then suspended in a dry, water immiscible organic solventcontaining polymer and release modifying compounds, and the suspensionsonicated to disperse the particles. Using this method, two formulationsof P3A were developed (TABLES 7 and 8). Notably, HPMCAS-MF was used asthe pore forming reagent as it is water insoluble at low pH (i.e., formsa gel), and become water soluble at high pH. At least 80% P3A activitywas recovered after dissolution of the P3A particles made using theseformulations as measured by the CENTA chromatogenic assay (TABLES 7 and8) (Bebrone et al., 2001; Antimicrobial Agents and Chemotherapy;45:1868).

TABLE 7 P3A formulation 1 Amt (g) in Component Item 500 ml % Total % APIP3A 2.50 0.5 9.77 Pore Former HPMCAS-MF 1.67 0.3 6.53 Matrix Aquacoat(FMC) 50.00 10.1 58.62 Lube Sodium-Stearyl 0.83 0.2 3.24 Fumarate BufferSodium Hydrogen 0.59 0.1 2.31 Phosphate Protectant Trehalose 5.00 1.019.54 Water 440.00 88.8 Total Water 489.85 Total Solids 25.59 100.00Solids in Matrix 30.00 Activity Recovered 82.00

TABLE 8 P3A formulation 2 Amt (g) in Component Item 500 ml % Total % APIP3A 11.25 2.30 39.37 Pore Former HPMCAS-MF 1.50 0.30 5.25 MatrixAquacoat (FMC) 50.00 10.00 52.49 Lube Sodium-Stearyl 0.33 0.10 1.14Fumarate Buffer Sodium Hydrogen 0.50 0.10 1.75 Phosphate ProtectantTrehalose 0.00 0.00 0.00 Water 437.50 87.50 Total Water 472.50 TotalSolids 28.58 100.00 Solids in Matrix 30.00 Activity Recovered 80.00

Another approach uses aqueous phases but no organic solvent. Here, theenzyme, buffer components, a polymer latex, and stabilizing andrelease-modifying excipients are dissolved/dispersed in water. Theaqueous dispersion is spray-dried, leading to coalescence of the latex,and incorporation of the protein and excipients in particles of thecoalesced latex. If the release modifiers are insoluble at acidicconditions but soluble at higher pHs (such as carboxylic acidic) thenrelease from the matrix should be inhibited in the gastric environment.

Formulation approaches are shown in FIGS. 25-28.

Example 6 GI Tract Localization of Beta-Lactamase Release

These studies are designed to identify preferred sites of beta-lactamasedelivery to the GI tract to achieve efficient antibiotic absorption andmicrobiome protection.

Beta-lactamase SYN-004 a/k/a P3A (resuspended in PBS or other buffer, orany of the formulations of SYN-004) is delivered directly to variousregions of the intestinal tract of dogs via intubation or a fistula inthe intestine. Animals receive oral antibiotic, such as amoxicillin, oramoxicillin/clavulanic acid (Augmentin), and P3A via direct delivery tothe small intestine including the duodenum, jejunum, ileum, and/orcecum. Plasma levels of the antibiotic are measured and the diversity ofthe microbiome is assessed by 16S sequence analysis of microbes in thestool, as an assessment of antibiotic degradation, and microbiomeprotection. Cohorts include antibiotic alone, antibiotic/inhibitoralone, antibiotic/inhibitor+P3A, and antibiotic+P3A, delivered to theindicated areas of the small intestine.

To perform this study, fistulas are implanted in groups of dogs (n=3-5per cohort) at the indicated locations in the small intestine including,the duodenum, jejunum, ileum, cecum, and ascending colon (Table 1). Thedogs receive a dosage of oral antibiotic, such as amoxicillin or anantibiotic/inhibitor combination, such as amoxicillin/clavulanic acid(Augmentin) as a single dose. P3A is delivered as an oral pill (usingthe current SYN-004 formulation) or dissolved in PBS buffer via directinfusion into the fistula and delivered within 30 minutes after the oralantibiotic. Plasma samples are drawn from the dogs at various timepoints to measure antibiotic levels in the blood as a measure ofantibiotic absorption. Fecal samples are collected from the animals tomeasure the level of excreted antibiotics and to assess the intestinalmicrobiome using 16S sequence analyses, as an additional assessment ofantibiotic degradation.

TABLE 9 Treatment of fistulated dogs with oral antibiotic/inhibitor ororal antibiotic and SYN-004 Cohort Oral Antibiotic P3A 1 None None 2Antibiotic None 3 Antibiotic/inhibitor None 4 None P3A - current oralformulation 5 Antibiotic and/or P3A - current oral formulationantibiotic/inhibitor combo 6 Antibiotic and/or P3A - via fistula toduodenum antibiotic/inhibitor combo 7 Antibiotic and/or P3A - viafistula to jejunum antibiotic/inhibitor combo 8 Antibiotic and/or P3A -via fistula to ileum antibiotic/inhibitor combo 9 Antibiotic and/orP3A - via fistula to cecum antibiotic/inhibitor combo 10 Antibioticand/or P3A - via fistula to ascending colon antibiotic/inhibitor combo

The results allow the demonstration that delivery of P3A to the smallintestine results in protection of the microbiome and does not affectantibiotic plasma levels. The study allows the identification ofpreferred sites of beta-lactamase delivery to the small intestine orcolon to achieve microbiome protection. Related studies with differentantibiotics and/or antibiotic/inhibitor combinations may be undertakento specify key locations in the intestinal tract.

Example 7 Evaluation of SYN-004 Delivered with Augmentin orSultamicillin to Pigs

This study evaluates use of an enteric-coated pellet formulation ofSYN-004, e.g. those described elsewhere herein, delivered with oralamoxicillin/clavulanate (Augmentin; antibiotic/inhibitor combination) oramoxicillin alone for protection of the microbiome without affectingantibiotic absorption.

SYN-004 enteric-coated pellet formulation, and oralamoxicillin/clavulanate (Augmentin) or oral amoxicillin alone, cohorts(n=3-5) of normal young pigs (˜50 lbs) are treated with clindamycin as apositive control for microbiome damage (one time), or oral Augmentin+/−SYN-004 for 5-7 consecutive days or oral amoxicillin+/−SYN-004 for5-7 consecutive days (TABLE 10). SYN-004 treatment is started 1 dayprior to oral antibiotic delivery. Plasma and stool is collected daily,beginning the day prior to treatment (Day −1). Plasma is monitored foramoxicillin levels and stool is subjected to 16S RNA sequencing tomonitor the diversity of the microbiome and stool is analyzed for thepresence of amoxicillin.

This study may display that at one or both of the Augmentin/SYN-004doses or the amoxicillin/SYN-004 doses, the plasma levels of amoxicillinare not affected while the microbiome is protected, indicating that theSYN-004 degraded the amoxicillin excreted into the intestine followingamoxicillin absorption without affecting the initial amoxicillinabsorption. Other orally-delivered antibiotics and/orantibiotic-inhibitor combinations are evaluated in an analogous manner.

TABLE 10 Treatment of normal pigs with oral antibiotic (Augmentin oramoxicillin) and oral SYN-004 Cohort (n = 3-5) Antibiotic Oral SYN-004 1none none 2 Clindamycin none (30 mg/kg) 3 Oral Augmentin or SYN-004amoxicillin High dose (875 mg/kg BID) (12.5 mg/kg QID) 4 Oral Augmentinor SYN-004 amoxicillin Low dose (875 mg/kg BID) (0.5 mg/kg QID) 5 OralAugmentin or none amoxicillin (875 mg/kg BID) 6 Oral Augmentin orSYN-004 amoxicillin High dose (500 mg/kg BID) (12.5 mg/kg QID) 7 OralAugmentin or SYN-004 amoxicillin Low dose (500 mg/kg BID) (0.5 mg/kgQID) 8 Oral Augmentin or none amoxicillin (500 mg/kg BID)

Evaluation of an enteric-coated pellet formulation of SYN-004's efficacyin the degradation of a covalently bound oral antibiotic/inhibitorcombination (sultamicillin) without affecting antibiotic absorption isundertaken. It is not clear if the ampicillin and/or sulbactamcomponents of sultamicillin are functional prior to metabolism (breakingof the ester bond linking the ampicillin and sulbactam). If theampicillin is inactive in sultamicillin, this would function as anadditional fail-safe where SYN-004 would not degrade sultamicillin priorto absorption, but is predicted to efficiently degrade ampicillin whenexcreted back into the intestine to protect the microbiome.

Using the SYN-004 enteric-coated pellet formulation described herein,oral sultamicillin (ampicillin/sulbactam 1:1 covalent linkage) or oralampicillin, cohorts (n=3-5) of normal young pigs (˜50 lbs) are treatedwith clindamycin as a positive control for microbiome damage (one time),or oral sultamicillin or oral ampicillin +/− SYN-004 for 5-7 consecutivedays (TABLE 11). SYN-004 treatment is started 1 day prior to oralantibiotic delivery. Plasma and stool is collected daily, beginning theday prior to treatment (Day −1). Plasma is monitored for ampicillinlevels and stool is subjected to 16S RNA sequencing to monitor thediversity of the microbiome, and stool is analyzed to measure ampicillinlevels. This study may display that at one or both of the oralsultamicillin/SYN-004 doses, and/or the oral ampicillin/SYN-004 dose,the plasma levels of ampicillin are not affected while the microbiome isprotected, indicating that the SYN-004 degraded the ampicillin excretedinto the intestine following ampicillin absorption without affecting theinitial ampicillin absorption.

TABLE 11 Treatment of normal pigs with oral sultamicillin or ampicillinand SYN-004 Cohort (n = 3-5) Antibiotic Oral SYN-004 1 none none 2Clindamycin none (30 mg/kg) 3 Oral Sultamicillin or SYN-004 ampicillinHigh dose (750 mg/kg BID) (12.5 mg/kg QID) 4 Oral Sultamicillin orSYN-004 ampicillin Low dose (750 mg/kg BID) (0.5 mg/kg QID) 5 OralSultamicillin or none ampicillin (750 mg/kg BID) 6 Oral Sultamicillin orSYN-004 ampicillin High dose (375 mg/kg BID) (12.5 mg/kg QID) 7 OralSultamicillin or SYN-004 ampicillin Low dose (375 mg/kg BID) (0.5 mg/kgQID) 8 Oral Sultamicillin or none ampicillin (375 mg/kg BID)

Example 8 In Vivo Evaluation of Modified-Release Formulations of P3A

The modified-release formulations of P3A, inclusive of those describedelsewhere herein, designed to release at preferred sites in the smallintestine as determined by the study outlined in Example 6, is tested inrodents, dogs, and/or pigs to determine if P3A is efficacious in thedegradation of an oral antibiotic/inhibitor combination or oralantibiotic alone, without affecting antibiotic absorption.

Results from Example 6 are expected to identify the preferred sites ofP3A delivery to the small intestine to achieve efficient oral antibioticabsorption with protection of the microbiome. Using the data fromExample 6, formulations of P3A with the chosen release profile areevaluated in rodents, pigs, and/or dogs as described in Examples 5 and7. Antibiotics chosen for initial evaluation include theantibiotic/inhibitor combinations of Augmentin, sultamicillin, and theantibiotics amoxicillin and/or ampicillin. For pig or dog studies,cohorts (n=3-5) of normal young pigs or beagle dogs are treated withclindamycin (once time) as a positive control for microbiome damage, ororal antibiotic/inhibitor (Augmentin, sultamicillin) or oral antibiotic(amoxicillin or ampicillin) +/−P3A for 5-7 consecutive days (TABLE 12).P3A treatment is started 1 day prior to oral antibiotic delivery. Plasmaand stool is collected daily, beginning the day prior to treatment (Day−1). Plasma is monitored for antibiotic levels and stool is subjected to16S RNA sequencing to monitor the diversity of the microbiome and stoolis analyzed for antibiotic levels. This study may display that at one orboth of the oral antibiotic/inhibitor/P3A doses, and/or at one or bothof the oral antibiotic/P3A doses, the plasma levels of antibiotic arenot affected while the microbiome is protected, indicating that the P3Adegraded the antibiotic excreted into the intestine following antibioticabsorption without affecting the initial antibiotic absorption.

TABLE 12 Treatment of normal pigs and/or dogs with modified-releaseformulations of P3A and oral antibiotic/inhibitor combinations(Augmentin, and/or sultamicillin) or oral antibiotic (amoxicillin and/orampicillin) Cohort Oral P3A (n = 3-5) Antibiotic (modified-releaseformulation) 1 none none 2 Clindamycin none (30 mg/kg) 3 Oral AntibioticP3A High Dose High dose (TBD mg/kg BID) (12.5 mg/kg QID) 4 OralAntibiotic P3A High Dose Low dose (TBD mg/kg BID) (0.5 mg/kg QID) 5 OralAntibiotic none High Dose (TBD mg/kg BID) 6 Oral Antibiotic P3A Low DoseHigh dose (TBD mg/kg BID) (12.5 mg/kg QID) 7 Oral Antibiotic P3A LowDose Low dose (TBD mg/kg BID) (0.5 mg/kg QID) 8 Oral Antibiotic none LowDose (TBD mg/kg BID)

Example 9 Evaluation of P3A as a Prophylactic to Prevent C. difficileDisease (CDI) Following Oral Antibiotic Treatment in Hamsters

These studies evaluate the efficacy of SYN-004 (current entericformulation or modified-release formulations of P3A, e.g. as describedherein) in the prevention of CDI in the hamster disease model.

SYN-004 or modified-release formulations of P3A are tested in rodentmodels of CDI. Rodent models include the Syrian Golden hamster(Mesocricetus auratus) C. difficile model (Sambol and Tang, 2001; J.Infect. Disease 183:1760). The hamster model has been referred to as“the gold standard” small animal model for the evaluation of theefficacy of a variety of prophylactic and therapeutic interventionsagainst CDI. CDI is induced in the hamsters using the followingprotocol. Male Golden Syrian hamsters, purchased from Harlan(Indianapolis, Ind.) are pretreated 5 days or 24 hours prior toinfection with a single subcutaneous injection of clindamycin at 10 or30 mg/kg to deplete the animal's microbiome and predispose them to C.difficile infection. As ampicillin is also a risk for C. difficileinfection (Freeman and Wilcox, 1999; Microbes Infect. 1:377), oralAugmentin, sultamicillin, ampicillin and/or amoxicillin is used in placeof clindamycin to predispose the animals to C. difficile infection.Plasma is collected at various times prior to and after antibioticdelivery to measure antibiotic blood levels. On the day of infection,animals are inoculated by oral gavage with 10⁶ C. difficile (ATCC 43255)vegetative cells per hamster. The C. difficile inoculum is prepared bygrowing the bacteria in Difco reinforced clostridial medium with 1%Oxyrase for 24 hrs under anaerobic conditions. The optical density at600 nm is adjusted to 1.5 and then diluted 1:10. The hamsters are given0.75 ml of this suspension orally via gavage. An aliquot of the inoculumis then serially diluted, plated on brucella agar supplemented withhemin and vitamin K₁ (Remel, Lenexa, Kans.), and incubated anaerobicallyfor 48 hrs in an airtight container (Pack-Anaero MGC) to determine theinfection titer. Animals are observed twice daily during the first 24hrs post-infection and then every 2 hrs for the following 24 hrs duringthe acute phase of the disease, followed by twice daily for theremainder of the study. Signs of CDI include signs of mortality andmorbidity, presence of diarrhea as indicated by a wet tail, and overallappearance including activity, general response to handling, touch, orruffled fur. Body weights are monitored every 2 to 3 days.

To evaluate the prophylactic potential of SYN-004 or modified-releaseformulations of P3A, it is administered orally beginning at the time oforal antibiotic administration, 1 day prior to C. difficile infection,and continued for the duration of the studies, up to 28 days. Disease iscompared in animals that receive clindamycin (as the positive control)or oral antibiotic/inhibitor combinations or oral antibiotics (OralAntibiotic). The efficacy of the P3A treatment groups are compared tocontrol animals that receive no treatment, animals that receive thestandard of care, vancomycin (20 mg/kg orally daily beginning 24 hrsafter infection and continued for 5 days), or animals that receive bothvancomycin and P3A. Plasma is monitored for antibiotic levels and stoolis subjected to 16S RNA sequencing to monitor the diversity of themicrobiome. Efficacy evaluations include mortality and evaluation of C.difficile bacteria titers and/or C. difficile toxins A and B in cecalcontents, at the time of death or at the end of the study followingeuthanasia. The results may show that treatment with the oralantibiotics and the P3A yeast at one or both doses, did not affect bloodlevels of the antibiotic and protected the animals from CDI, indicatingthat the P3A expressed by the yeast degraded the antibiotic excretedinto the intestine following antibiotic absorption without affecting theinitial antibiotic absorption. See TABLE 13 for the experimental design.

TABLE 13 C. difficile efficacy hamster study treatment groups Cohort C.diff (n = 6-10) Antibiotic inoculation Treatment 1 none None none 2Clindamycin + none (30 mg/kg) 3 Oral Antibiotic + none Dose TBD 4 OralAntibiotic + vancomycin Dose TBD 5 Oral Antibiotic + SYN-004 or P3A DoseTBD High dose (12.5 mg/kg QID) 6 Oral Antibiotic + SYN-004 or P3A DoseTBD Low dose (0.5 mg/kg QID) 7 Oral Antibiotic + Vancomycin + SYN-004Dose TBD (or P3A) Low dose (0.5 mg/kg QID)

Example 10 Evaluation of P3A as a Prophylactic to Prevent C. difficileDisease (CDI) Following Oral Antibiotic Treatment in Pigs

These studies evaluate the efficacy of SYN-004 (enteric formulation ormodified-release formulations of P3A, e.g. as described herein) in theprevention of CDI in humanized pigs.

SYN-004 or modified-release formulations of P3A are tested in ahumanized pig model of CDI. The humanized pig model is a model of thehuman gastrointestinal tract where the gnotobiotic pigs arereconstituted with human fecal homogenates (Zhang et al., Gut Microbes4:193). The humanized pigs are treated with antibiotics (clindamycin,Augmentin, sultamicillin, ampicillin or amoxicillin) to disrupt theirintestinal microbiome and then exposed to C. difficile after which theydevelop CDI including C. difficile associated diarrhea (CDAD).

To test the prophylactic potential of SYN-004 or modified-releaseformulations of P3A, P3A is administered one day prior to antibiotictreatment (Day −1), and maintained for the duration of the antibiotictreatment. Clindamycin is delivered 1 to 5 days prior to C. difficileinoculation. Oral antibiotics such as Augmentin, sultamicillin,ampicillin or amoxicillin, are delivered beginning 1 to 5 days prior toC. difficile inoculation, and maintained for 5-7 days. The antibioticsare used to disrupt the intestinal microbiome to predispose the animalsto C. difficile infection. Plasma levels of antibiotics are monitoredprior to antibiotic treatment, and during treatment to assess antibioticabsorption. C. difficile vegetative cells or spores are administered, atdoses ranging from 10⁶ to 10⁸, and animals are monitored for CDIsymptoms including CDAD. Animals exposed to C. difficile are expected todevelop disease symptoms within 48 hrs of bacterial inoculation (Steeleet al., 2010; J. Infect. Dis 201:428). CDI is compared in animals thatreceive clindamycin or oral antibiotics, such as Augmentin,sultamicillin, ampicillin or amoxicillin (Oral Antibiotic). The efficacyof the P3A treatment groups are compared to control animals that receiveno treatment, animals that receive the standard of care, vancomycin (20mg/kg orally daily beginning 24 hrs after infection and continued for. 5days), or animals that receive both vancomycin and P3A. The results mayshow that treatment with the oral antibiotics and the oral P3A at one orboth doses, did not affect blood levels of the antibiotic and protectedthe animals from CDI, indicating that the P3A degraded the antibioticexcreted into the intestine following antibiotic absorption withoutaffecting the initial antibiotic absorption. See TABLE 14 for theexperimental design.

TABLE 14 SYN-004 or modified-release formulations of P3A C. difficileefficacy humanized pig study treatment groups Cohort C. diff (n = 2-3)Antibiotic inoculation Treatment 1 none None none 2 Clindamycin + none(30 mg/kg) 3 Oral Antibiotic + none Dose TBD 4 Oral Antibiotic +vancomycin Dose TBD 5 Oral Antibiotic + SYN-004 or P3A Dose TBD Highdose (12.5 mg/kg QID) 6 Oral Antibiotic + SYN-004 or P3A Dose TBD Lowdose (0.5 mg/kg QID) 7 Oral Antibiotic + Vancomycin + SYN-004 Dose TBD(or P3A) Low dose (0.5 mg/kg QID)

Example 11 Evaluation of SYN-004 and Oral Antibiotics in an ArtificialSmall and Large Intestine System

The artificial small and large intestine system, TIM and/or TIM2 (see,e.g. Yoo, J. Y., & Chen, X. D. (2006). GIT physicochemical modeling—ACritical Review, International Journal of Food Engineering, 2(4), thecontents of which are hereby incorporated by reference), is used toevaluate the current, enteric-coated SYN-004 formulation and/ormodified-release formulations of SYN-004, e.g. as described herein, tomore specifically localize the site(s) of SYN-004 release and antibioticrelease within the intestinal track.

Example 12 Genetically-Modified Yeast for Delivery of P3A to theIntestinal Tract

Genetically-modified microorganisms are tested as delivery vehiclesto-administer P3A to the intestinal track to protect the microbiomewhile not affecting antibiotic absorption and therefore, antibioticefficacy.

Yeast genetically-modified to produce the antibiotic-degrading enzyme,P3A, are produced similarly to that described for the C. difficiletoxin-binding proteins in “Methods and Compositions for InhibitingClostridium difficile” filed Nov. 4, 2014, Ser. No. 62/074,993, thecontents of which are hereby incorporated by reference in theirentirety. Briefly, the P3A coding region is codon optimized forexpression in the yeast, S. cerevisiae, modified to reduced DNAhomologies, and evaluated for the presence of N-linked glycosylationsites, synthesized and cloned into the yeast expression plasmid, pD1214(DNA 2.0) that contains the strong, constitutive TEF promoter, and aselectable URA3+ marker. Different S. cerevisiae leader sequences thatfacilitate secretion are known and are utilized to mediate P3Asecretion. A series of S. cerevisiae secretion vectors are availablewhich contain a panel of different leader sequences to facilitatesecretion. An exemplary secretion signal is the yeast mating factoralpha (MAT alpha) signal, which is a 89 amino acid sequence composed ofthe signal and the prosequence which is cleaved in the Golgi by Kex2, anendogenous yeast protease, to yield the mature, secreted protein. Theinvertase and other signal sequences are naturally cleaved duringtranslocation and secretion of the protein by signal peptidase and donot require additional protease cleavage steps.

At least two strategies may be used to generate S. cerevisiae, substrainboulardii, transformants that secrete P3A. One strategy is theproduction of a S. boulardii URA3 knockout strain to allow the use ofthe P3A expression plasmids that contain the URA3 selectable marker togenerate transformants (non-integrated, containing the plasmids) to usein efficacy evaluation in rodents and/or pigs. The S. boulardii URA3knockout is generated using the CRISPR recombination system (DiCarlo etal, 2013, Nucleic Acids Res. 41:4436). The S. boulardii strain,designation Sb48 (ATCC Product #MYA-796) submitted to ATCC by D. A.Stevens (McCullough et al., 1998; J. Clinical Microbiology, 36:2613) isused for these studies. Three potential wild-type Cas9 cleavage sites inthe upstream region of the URA3 gene are identified and approximately500 pb of the regions surrounding these target sites are sequenced toensure the presence of the sites in this yeast strain. A homologyconstruct is designed that contains an approximate 10 bp region in themiddle replaced by an insert that contains multiple stop codons in allframes ensuring that the first stop codon is in the URA3 reading frame.The CRISPR system is used to create the recombination/insertion and theURA3-clones are selected on FOA (5-fluoroorotic acid) media. 5-FOAallows the selection for URA3-mutants, as an active URA3 gene (encodesorotidine 5′-phosphate decarboxylase) converts FOA into a toxic compoundcausing cell death. The selected clones are then tested to ensure thatthey will not grow on media without uracil. Selected clones aresequenced to verify the expected integration. Once the S. boulardiistrain is confirmed to be URA3-, the yeast are transformed with the P3Aencoding plasmids. Clones are identified by plating on media withouturacil. The resulting transformants are screened for secretion of P3Ausing SDS/PAGE. Filtered yeast supernatants are evaluated for activityusing the CENTA beta-lactamase biological activity assay (Bebrone etal., 2001; Antimicrobial Agents and Chemotherapy., 45:1868).

A second strategy generates stable integrants in the wild-type S.boulardii strain using a neomycin resistance gene (neo) as theselectable marker. Without neo expression, S. boulardii is sensitive toG418. The S. boulardii strain, designation Sb48 (ATCC Product #MYA-796)submitted to ATCC by D. A. Stevens (McCullough et al., 1998; J. ClinicalMicrobiology, 36:2613) is used for these studies. Integration regionsare chosen based on Flagfeldt et al (2009, Yeast 26:545), wherechromosomal integration sites were screened for high level heterologousgene expression. The integration sites that show the highest expressionlevels, Regions 20, 21, and 19 are sequenced in the wild-type S.boulardii strain to verify their presence. Once verified, a region ischosen and plasmids containing integration cassettes are designed. Theintegration cassettes containing the P3A expression cassette, a neoexpression cassette, at least 500 bp of homology sequence from theupstream part of the integration region and at least 500 bp of homologysequence from the downstream part of the integration region so that theintegration region is deleted via the homologous recombination event.The wild-type S. boulardii is transformed with the integration cassettesand clones are selected for G418 resistance. Clones are picked, culturesgrown, and supernatants screened for the presence of the P3A protein viaSDS/PAGE. Filtered yeast supernatants are evaluated for biologicalactivity using the CENTA beta-lactamase biological activity assay(Bebrone et al., 2001; Antimicrobial Agents and Chemo., 45:1868). Clonesare chosen, based on protein expression levels and biological activity,and the insert is sequenced to verify the integrity of the integratedsequence.

The P3A-expressing yeast are tested in a rodent, pig, and/or dogmodel(s) to determine if the P3A-expressing yeast are efficacious in thedegradation of an oral antibiotic/inhibitor combination or oralantibiotic alone, without affecting antibiotic absorption. For pig ordog studies, cohorts (n=3-5) of normal young pigs or beagle dogs aretreated with clindamycin (once time) as a positive control formicrobiome damage, or oral antibiotic/inhibitor (Augmentin orsultamicillin) or oral antibiotic (amoxicillin and/or ampicillin), for5-7 consecutive days (TABLE 15). P3A-expressing yeast are delivered BIDstarting 3 days prior to antibiotic treatment and maintained throughoutthe antibiotic treatment period. Plasma and stool is collected daily,beginning the day prior to yeast treatment (Day −4) and prior to oralantibiotic treatment (Day −1). Plasma is monitored for antibiotic levelsand stool is subjected to 16S RNA sequencing to monitor the diversity ofthe microbiome. The results may show that one or both of theantibiotic/P3A yeast doses, the plasma levels of antibiotic are notaffected while the microbiome is protected, indicating that the P3Aexpressed by the yeast degraded the antibiotic excreted into theintestine following antibiotic absorption without affecting the initialantibiotic absorption.

TABLE 15 Treatment of normal pigs and/or dogs with S. boulardiiexpressing P3A and an oral antibiotic/inhibitor (Augmentin orsultamicillin) or an oral antibiotic (amoxicillin and/or ampicillin)Cohort Oral (n = 3-5) Antibiotic S. boulardii 1 none none 2 Clindamycinnone (30 mg/kg) 3 Oral Antibiotic S. boulardii wt High dose 3 × 10¹⁰ cfuBID (TBD mg/kg BID) 4 Oral Antibiotic S. boulardii High dose P3Aexpressing (TBD mg/kg BID) 3 × 10¹⁰ cfu BID 5 Oral Antibiotic none Highdose (TBD mg/kg BID) 6 Oral Antibiotic S. boulardii wt Low Dose 3 × 10¹⁰cfu BID (TBD mg/kg BID) 7 Oral Antibiotic S. boulardii Low Dose P3Aexpressing (TBD mg/kg BID) 3 × 10¹⁰ cfu BID 8 Oral Antibiotic none LowDose (TBD mg/kg BID)

Example 13 In Vivo Analysis of Yeast-Expressed P3A in Hamster CDI Model

P3A-expressing yeast are evaluated for the prevention of C. difficileinfection and disease in a hamster model of C. difficile disease.

The S. boulardii transformants expressing the P3A are evaluated inrodent models of C. difficile disease (CDI), including the Syrian Goldenhamster (Mesocricetus auratus) C. difficile model (Sambol and Tang,2001; J. Infect. Disease 183:1760) as described in Example 9.

To evaluate the prophylactic potential of the S. boulardii transformantsexpressing P3A, the yeast are administered, via oral gavage, at dosesranging from 100 to 500 mg, approximately 2×10⁸ to 2×10¹⁰ cfu/animaldaily beginning at the time of antibiotic administration, 5 or 1 dayprior to C. difficile infection, and continued for the duration of thestudies, up to 28 days. As yeast are not sensitive to antibiotics, theyeast will remain viable even in the presence of antibiotics. Disease iscompared in animals that receive clindamycin or oral Augmentin,sultamicillin, ampicillin, or amoxicillin (Oral Antibiotic). Plasma ismonitored for antibiotic levels and stool is subjected to 16S RNAsequencing to monitor the diversity of the microbiome. The efficacy ofthe P3A-expressing yeast are compared to control animals that receive notreatment, animals that receive the standard of care, vancomycin (20mg/kg orally daily beginning 24 hrs after infection and continued for 5days), or animals that receive both vancomycin and the yeast. Efficacyevaluations include mortality and evaluation of C. difficile bacteriatiters and/or C. difficile toxins A and B in cecal contents, at the timeof death or at the end of the study following euthanasia. The resultsmay show that treatment with the oral antibiotics and the P3A yeast atone or both doses, did not affect blood levels of the antibiotic andprotected the animals from CDI, indicating that the P3A expressed by theyeast degraded the antibiotic excreted into the intestine followingantibiotic absorption without affecting the initial antibioticabsorption. See TABLE 16 for the experimental design.

TABLE 16 P3A-expressing yeast C. difficile efficacy hamster studytreatment groups Cohort C. diff (n = 6-10) Antibiotic inoculationTreatment 1 none None none 2 Clindamycin + none (30 mg/kg) 3 OralAntibiotic + none Dose TBD 4 Oral Antibiotic + vancomycin Dose TBD 5Oral Antibiotic + wt yeast Dose TBC High dose (10¹⁰ cfu BID) 6 OralAntibiotic + P3A yeast Dose TBD High dose (10¹⁰ cfu BID) 7 OralAntibiotic + P3A yeast Dose TBD Low dose (10⁸ BID) 8 Oral Antibiotic +Vancomycin + P3A yeast Dose TBD High dose (10¹⁰ cfu BID)

Example 14 In Vivo Analysis of Yeast-Expressed P3A in Porcine Model ofCDI

Studies of the use of P3A-expressing yeast in the prevention of C.difficile infection and disease in a humanized pig model of C. difficiledisease are undertaken.

The S. boulardii transformants expressing the P3A are tested in ahumanized pig model of CDI. The humanized pig model is described inExample 10. The humanized pigs are treated with antibiotics (clindamycinor Augmentin, sultamicillin, ampicillin, or amoxicillin) to disrupttheir intestinal microbiome and then exposed to C. difficile after whichthey develop CDI including C. difficile associated diarrhea (CDAD).

To test the prophylactic potential of P3A-expressing yeast, the yeastare administered one day prior to antibiotic treatment (Day −1), anddelivered BID for the duration of the antibiotic treatment. Yeast aregiven at doses ranging from 250 mg to 3000 mg/animal, approximately5×10⁹ to 6×10¹⁰ cfu/animal. Clindamycin is delivered 1 to 5 days priorto C. difficile inoculation. Oral Augmentin, sultamicillin, ampicillin,or amoxicillin (Oral Antibiotic) is delivered beginning 1 to 5 daysprior to C. difficile inoculation, and maintained for 5-7 days. Theantibiotics are used to disrupt the intestinal microbiome to predisposethe animals to C. difficile infection. C. difficile vegetative cells orspores are administered, at doses ranging from 10⁶ to 108, and animalsare monitored for CDI symptoms including CDAD. CDI is compared inanimals that receive clindamycin or Augmentin, sultamicillin,ampicillin, or amoxicillin (Oral Antibiotic). Plasma is monitored forantibiotic levels and stool is subjected to 16S RNA sequencing tomonitor the diversity of the microbiome. The efficacy of theP3A-expressing yeast treatment groups are compared to control animalsthat receive no treatment, animals that receive the standard of care,vancomycin (20 mg/kg orally daily beginning 24 hrs after infection andcontinued for 5 days), or animals that receive both vancomycin andP3A-expressing yeast. The results may show that treatment with the oralantibiotics and the P3A yeast at one or both doses, did not affect bloodlevels of the antibiotic and protected the animals from CDI, indicatingthat the P3A expressed by the yeast degraded the antibiotic excretedinto the intestine following antibiotic absorption without affecting theinitial antibiotic absorption. See TABLE 17 for the experimental design

TABLE 17 P3A-expressing yeast C. difficile efficacy pig study treatmentgroups Cohort C. diff (n = 2-3) Antibiotic inoculation Treatment 1 noneNone none 2 Clindamycin + none (30 mg/kg) 3 Oral Antibiotic + none DoseTBD 4 Oral Antibiotic + vancomycin Dose TBD 5 Oral Antibiotic + Wt yeastDose TBD High dose (3 × 10¹⁰ cfu BID) 6 Oral Antibiotic + P3A yeast DoseTBD High dose (3 × 10¹⁰ cfu BID) 7 Oral Antibiotic + P3A yeast Dose TBDLow dose (2.5 10⁹ BID) 8 Oral Antibiotic + Vancomycin + P3A yeast DoseTBD High dose (3 × 10¹⁰ cfu BID)

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

REFERENCES

The following are hereby incorporated by reference in their entireties:

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1.-64. (canceled)
 65. A modified-release formulation comprising abeta-lactamase, wherein the formulation releases a substantial amount ofthe beta-lactamase in the gastrointestinal (GI) tract, and wherein theformulation comprises at least one modified-release pellet with eachpellet comprising: about 10-20% by weight beta-lactamase; about 10-20%by weight sucrose sphere; about 20-30% by weight hydroxypropylcellulose;about 10-20% by weight a first enteric polymer; about 20-30% by weight asecond enteric polymer; about 1-10% by weight triethyl citrate; andabout 1-2% by weight buffer salt; and wherein the formulation releasesthe beta-lactamase at a pH of greater than 6.7.
 66. The modified-releaseformulation of claim 65, wherein the first enteric polymer is EUDRAGITL100.
 67. The modified-release formulation of claim 65, wherein thesecond enteric polymer is EUDRAGIT S100.
 68. (canceled)
 69. A method ofpreventing an antibiotic-associated adverse effect in a subject in needthereof, comprising administering an effective amount of themodified-release formulation of claim 65, and wherein the antibiotic isan oral antibiotic, the oral antibiotic being a substrate for thebeta-lactamase; and wherein the subject is undergoing treatment with theoral antibiotic.
 70. The method of claim 69, wherein theantibiotic-associated adverse effect is Clostridium difficile infection.71. The method of claim 69, wherein the antibiotic-associated adverseeffect is antibiotic associated diarrhea.
 72. The method of claim 69,wherein the beta-lactamase comprises an amino acid sequence of SEQ IDNO: 1, having asparagine (N) at position 276, according to Amblerclassification (P3A). 73.-74. (canceled)
 75. The modified-releaseformulation of claim 65, wherein the beta-lactamase comprises an aminoacid sequence of SEQ ID NO: 1, having asparagine (N) at position 276,according to Ambler classification (P3A).
 76. The modified-releaseformulation of claim 65, wherein the formulation comprises at least onemodified-release pellet with each pellet comprising: about 13% by weightbeta-lactamase comprising an amino acid sequence of SEQ ID NO: 1, havingasparagine (N) at position 276, according to Ambler classification(P3A); about 18% by weight sucrose sphere; about 28% by weighthydroxypropylcellulose; about 12% by weight EUDRAGIT L100 about 25% byweight EUDRAGIT S100; about 4% by weight triethyl citrate; and about 1%by weight buffer salt; and wherein the formulation releases thebeta-lactamase at a pH of greater than 6.7.
 77. The method of claim 69,comprising administering an effective amount of the modified-releaseformulation of claim 76.